Anti-gd2 antibodies

ABSTRACT

In this application are described chimeric, humanized, affinity matured, stability enhanced, and bispecific Anti-GD2 antibodies and fragments thereof. Also provided are methods of using individual antibodies or compositions thereof for the detection, prevention, and/or therapeutical treatment of GD2-related diseases, in particular, neuroblastoma.

INTRODUCTION

Monoclonal antibody (MoAb) therapy is an accepted treatment modality forcancers, with five MoAbs having received FDA approval for solid tumorsin adults, including colorectal and breast cancer, non small cell lungcancer, squamous cell carcinoma, and melanoma (Boyiadzis et al., 2008,Expert Opin Biol Ther 8, 1151-8; Yan et al., 2008, Cancer J 14, 178-83).This modality, however, has remained inadequately exploited for thetreatment of pediatric cancers. Unlike chemotherapy or radiation, MoAbtherapy is not myelosuppressive and genotoxic, generally with few longterm toxicities. These are critical considerations for young children.More importantly, MoAb is effective against metastatic cancer in blood,bone marrow and bone, typically found in high risk neuroblastoma (NB).As a class of agents, the pharmacokinetics and toxicities of human orhumanized IgG1 antibodies have been extensively studied. In addition,antibodies can carry cytotoxic payloads, whether immune based,radioisotopes, toxins or enzymes, thereby increasing the options fortargeted therapy.

Neuroblastoma (NB) is the most common extracranial solid tumor ofchildhood. In ˜50% of cases, curative strategies must tackle both softtissue mass and metastases in the bone marrow (BM). Dose-intensivechemotherapy improves tumor resectability, and post-surgical irradiationreduces the risk of relapse in the primary site to <10% (Kushner et al.,2001, J Clin Oncol 19, 2821-8). However, BM disease, as evidenced byhistology or metaiodobenzylguanidine (MIBG) scan, often persists andforebodes a lethal outcome (Matthay et al., 2003, J Clin Oncol 21,2486-91; Schmidt et al., 2008, Eur J Cancer 44, 1552-8). In addition,osteomedullary relapse is common, despite achieving near completeremission after induction therapy. Attempts at treatment intensificationhave met with both acute and long-term side effects, both of graveconcern for young patients. There is a scarcity of promising new agents,and to date, few if any target/pathway-specific small molecules haveshown major clinical benefit in patients with NB, although manypromising leads continue to accumulate. With a cure rate of <30% attoxicity limits among stage 4 patients diagnosed at >18 months of age,there is substantial room for improvement (Pearson et al., 2008, LancetOncol 9, 247-56).

Several factors make NB well suited for MoAb targeted immunotherapy.First, MoAb mediates highly efficient antibody-dependent cellularcytotoxicity (ADCC) of NB in the presence of human white cells. Second,MoAb induces complement-mediated cytotoxicity (CMC) of NB cells, whichlack decay accelerating factor CD55 (Cheung et al., 1988, J Clin Invest81, 1122-8) and homologous restriction factor CD59 (Chen et al., 2000,Cancer Res 60, 3013-8). Complement deposition on NB cells enhances ADCCthrough activation of the iC3b receptor on neutrophils (Kushner andCheung, 1992, Blood 79, 1484-90, Metelitsa et al., 2002, Blood 99,4166-73), available even after dose-intensive or myeloablativechemotherapy plus stem cell transplantation, if colony stimulatingfactors are given (Mackall, C L, 2000, Stem Cells 18, 10-8). Third, theuse of intensive chemotherapy (standard of care for NB) to achieveclinical remission causes prolonged lymphopenia and immunosuppression(Mackall et al., 2000, Blood 96, 754-762), such that patients are lesslikely to reject murine, chimeric or humanized MoAbs (Kushner et al.,2007, Pediatr Blood Cancer 48, 430-4).

GD2 is a disialoganglioside abundant on tumors of neuroectodermalorigin, including neuroblastoma and melanoma with highly restrictedexpression in normal tissues. At least two antibody families have beentested clinically, i.e. 3F8 (Cheung et al., 1985, Cancer Res 45, 2642-9)and 14.18 (Mujoo et al., 1989, Cancer Res 49, 2857-61). Chimeric ch14.18consists of the variable region of murine MoAb 14.18 and the constantregions of human IgG1-K (Gillies et al., 1989, J Immunol Methods 125,191-202). It demonstrates ADCC and CMC of NB and melanoma cells in vivo(Barker et al., 1991, Cancer Res 51, 144-9; Barker and Reisfeld, 1993,Cancer Res. 52, 362-7; Mueller et al., 1990, PNAS USA 87, 5702-05 0.Based on encouraging clinical responses in phase I studies, ch14.18 wastested in large phase II studies as consolidation therapy for stage 4 NB(German NB90 and NB97 studies). For the 166 patients >12 months atdiagnosis, even though event-free survival (EFS) was similar in patientsreceiving ch14.18 when compared to patients on maintenance chemotherapy,overall survival (OS) was improved, and the rate of BM relapse reducedin patients treated with ch14.18 (Simon et al., 2004, J Clin Oncol 22,3549-57). In 2001, the Children's Oncology Group (COG) initiated arandomized phase III trial to study the efficacy of the combination ofch14.18 with GMCSF and IL-2 in preventing NB relapse in patients incomplete remission (CR) after autologuous stem-cell transplantation(ASCT) (ClinicalTrials.gov NCT00026312) (Gilman et al., J Clin Oncol27:85-91, 2009), where a significant improvement in progression freesurvival (PFS) and OS at 2 years was found (Yu et al., N Engl J Med363:1324-1334, 2010). 3F8, a murine IgG3 MoAb specific for GD2, inducescell death, and mediates efficient ADCC and CMC against NB in vitro(Cheung et al., 2007, supra). Among patients with chemoresistant marrowdisease despite dose-intensive induction plus an aggressive salvageregimen, 80% achieved BM remission usually after 1 to 2 cycles of 5-dayantibody plus GM-CSF therapy (Kushner et al., 2007, Proc Amer Soc ClinOncol 25, 526s). Given the activity of m3F8 against chemoresistantmarrow disease, the use of m3F8 was expanded to patients in their firstremission with encouraging results. These favorable clinical outcomes inchildren could be improved if m3F8 is given as maintenance therapy overthe first 3-5 years of highest recurrence risk. However, humananti-mouse antibody response (HAMA) is a limiting factor when the immunesystem recovers when chemotherapy is finished. One strategy to reduceHAMA is to chimerize or humanize 3F8.

Therefore, there is a need for a chimeric and/or humanized 3F8 antibodyable to bind GD2 with high affinity, superior to m3F8, and able tomediate antibody-dependent cellular cytotoxicity thereby allowing longterm antibody therapy in order to reduce disease recurrence.

Herein described is the engineering and isolation of chimeric 3F8(ch3F8-IgG1) and humanized 3F8 (hu3F8-IgG1 and hu3F8-IgG4). Theseantibodies were made using standard recombinant methods, and selectedfor high expression by CHO-DG44 cell lines in serum free medium. Aspecial glycoform of hu3F8-IgG1n (also called hu3F8-IgG1-MAGE1.5) wasproduced using CHO-Mage1.5 cell line. This special glycoform has nofucose, and only terminal mannose and N-acetylglucose or glucose.Measured using surface Plasmon resonance used by Biacore systems,chimeric 3F8 and humanized 3F8 maintained a K_(D) similar to that ofm3F8. In contrast to other anti-GD2 antibodies, m3F8, ch3F8-IgG1,ch3F8-IgG4, hu3F8-IgG1, hu3F8-IgG4 and hu3F8-IgG1n had substantiallyslower k_(off), which translated into a slower wash off in vitro. Likem3F8, both chimeric and humanized 3F8 inhibited cell growth in vitro,not typical for other anti-GD2 antibodies. Similar measurementsindicated that chimeric and humanized IgG1 antibodies have morefavorable K_(D) compared to 14.G2a. Both blood mononuclear cell(PBMC)-ADCC and neutrophil (PMN)-ADCC of ch3F8-IgG1, hu3F8-IgG1, andhu3F8-IgG1n were superior (10 to >1000 fold) to that of m3F8, while CMCwas inferior. This superiority was consistently observed in ADCC assays,irrespective of donors or if NK92 transfected with human CD16 or CD32were used as killers. For CD16 mediated ADCC, hu3F8-IgG1n was 10-40 foldbetter compared to hu3F8-IgG1. PBMC-ADCC and CMC activity was greatlyreduced with hu3F8-IgG4 when compared to m3F8. Crossreactivity withother gangliosides were similar to that of m3F8. Using ¹³¹I labeledantibodies in biodistribution, hu3F8 forms had tumor to normal tissueratios comparable to those of m3F8. Hu3F8-IgG1 showed superioranti-tumor effect against NB xenografts when compared to m3F8.

Also described herein are novel hu3F8 antibodies, hu3F8H3L3, which havebeen designed to have enhanced stability profiles using anexperimentally derived crystal structure of m3F8 in combination withcomputational analysis using force methods.

SUMMARY OF THE INVENTION

Humanizing strategies share the premise that replacement of amino acidresidues that are characteristic of murine sequences with residues foundin the correspondent positions of human antibodies will reduce theimmunogenicity in humans of the resulting antibody in humans. However,replacement of sequences between species usually results in reduction ofantibody binding to its antigen, and loss of affinity. The art ofhumanization therefore lies in balancing replacement of the originalmurine sequence to reduce immunogenicity with the need for the humanizedmolecule to retain sufficient antigen binding to be therapeuticallyuseful.

The present invention provides engineered and isolated chimeric andhumanized 3F8 antibodies, having at least one CDR sequence derived fromthe m3F8, as well as antibody compositions, glycoforms of the antibody,antibodies with enhanced stability, antibodies with enhanced binding toFc receptors, antibodies with enhanced affinity to GD2, bispecificantibodies engineered to express a second distinct binding site or abispecific T-cell engager, or use of the Fv fragments of any of theantibodies of the present invention in modular IgG construction forbispecific, bispecific T-cell engaging (BiTE) antibodies, trispecific ormultispecific antibodies. These antibodies and encoding or complementarynucleic acids, vectors, host cells, compositions, formulations, devices,transgenic animals, transgenic plants related thereto, and methods ofmaking and using thereof, as described and enabled herein, incombination with what is known in the art are part of the presentinvention. The hu3F8-IgG1 antibody of the invention has significantlymore PMN-ADCC and PBMC-ADCC activities than m3F8 or any other knownanit-GD2 antibodies (including 14G.2a, ch14.18 or ME361), with intactcomplement mediated cytotoxicity (CMC), although less than m3F8. Thissuperiority was consistently observed in ADCC asays irrespective ofdonors or if NK92 transfected with human CD16 or CD32 were used askillers. This was important since ADCC is the proven mechanism foranti-tumor effects of MoAb in patients in general. Both hu3F8-IgG1 andhu3F8-IgG1n, a glycoform of hu3F8-IgG1, showed superior anti-tumoreffect against neuroblastoma NB) xenografts when compared with m3F8. TheIgG4 form of the antibody showed reduced effector function and iseffective for blocking pain side effects of anti-GD2 antibody therapy.Hu3F8-IgG1-DEL with a triple mutation in the heavy chain (orS239D/I332E/A330L) showed increased affinity to the Fc receptor (FcR).Analysis of 3F8:GD2 interaction resulted in design of a 3F8 antibodywith mutation in the heavy chain producing huH1I-gamma-1 andhuH3I-gamma-1 heavy chains thermodynamically calculated to conferincreased affinity to GD2.

The present invention provides at least one isolated engineeredchimeric, ch3F8, or humanized, hu3F8, antibody as described herein. Theantibody according to the present invention includes any protein orpeptide molecule that comprises at least one complementarity determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, derived from m3F8, in combination with a heavy chain or lightchain constant region, a framework region, or any portion thereof, thatcan be incorporated into an antibody of the present invention. In oneembodiment the invention is directed to a hu3F8 antibody comprising alight chain and a heavy chain described herein, each of the chainscomprising at least part of a human constant region and at least part ofa variable region derived from m3F8 which has specificity to GD2, saidantibody binding with high affinity to GD2 and mediating a desiredeffect, e.g. inhibiting cell growth in vitro, blocking pain side effectsdue to anti-GD2 antibody therapy, to name a few. The invention alsoincludes fragments or a derivative of such an antibody, such as one ormore portions of the antibody chain, such as the heavy chain constant,joining, diversity or variable regions, or the light chain constant,joining or variable regions. The antibodies can be of any class such asIgG, IgM, or IgA or any subclass such as IgG1, IgG2a, IgG4, and othersubclasses known in the art. Antibodies useful in the present inventionalso include antigen-binding antibody fragments of the antibodies of thepresent invention including, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked or disulfide-stabilized Fvs (sdFv or dsFv). Theinvention also includes single-domain antibodies comprising either a VLor VH domain. Further, the antibodies can be produced by any method,such as phage display, or produced in any organism, egg, or cell line,including bacteria, insect, yeast (fungi), mammal or other type of cellor cell line which produces antibodies with desired characteristics,such as humanized antibodies. The antibodies can also be formed bycombining a Fab portion and a Fc region from different species, or bykeeping the complementarity-determining regions and modifying theframework regions to that of another species.

The antibody can comprise at least one specified portion of at least onecomplementarity determining region (CDR) (e.g., CDR1, CDR2 or CDR3 ofthe heavy or light chain variable region) derived from a m3F8 orhu3F8(as such terms are defined herein), and/or at least one constant orvariable framework region or any portion thereof. The antibody aminoacid sequence can further optionally comprise at least one specifiedsubstitution, insertion or deletion as described herein or as known inthe art.

Preferred antibodies of the present invention include ch3F8-IgG1,ch3F8-IgG4, hu3F8-H1L1-IgG1, hu3F8-H2L2-IgG1, hu3F8-H1L2-IgG1,hu3F8-H2L1-IgG1, hu3F8-IgG4, hu3F8IgG1n, Hu3F8-IgG1-DEL, hu3F8H1L1S(hu3F8-IgG1 light chain interface enhanced), hu3F8H3L3 (hu3F8-IgG1 heavyand light chain stability enhanced), hu3F8H3L3S (interface and stabilityenhanced), hu3F8H1-I-gamma-1 (hu3F8-IgG1 affinity enhanced),hu3F8H3-I-gamma-1 (hu3F8-IgG1 stability and affinity enhanced) as wellas fragments and regions thereof.

In one embodiment, the present invention relates to chimeric monoclonalantibodies having binding specificity for GD2, wherein the antibodych3F8-IgG1 comprises heavy chain domain SEQ ID NO:1, and a light chainvariable domain SEQ ID NO:2. In another embodiment, the antibodych3F8-IgG4 comprises the heavy chain variable domain of SEQ ID NO:3 anda light chain domain of SEQ ID NO:2. Such chimeric antibodies arederived from murine 3F8 antibody, in certain embodiments, the chimericantibodies comprise a heavy chain whose complementarity determiningregions (CDR) 1, 2 and 3 are defined by amino acid residues 31-35, 50-64and 98-108, respectively, of SEQ ID NO:1. In certain embodiments, thechimeric antibodies comprise a light chain whose CDRs 1, 2 and 3 aredefined by amino acid residues 24-34, 50-56 and 89-95, respectively, ofSEQ ID NO:2. In certain embodiments, the chimeric antibodies comprise aheavy chain whose framework regions (FR) 1, 2, 3 and 4 are defined byamino acid residues 1-30, 36-49, 65-97 and 109-120, respectively, of SEQID NO:1. In certain embodiments, the chimeric antibodies comprise alight chain whose framework regions (FR) 1, 2, 3 and 4 are defined byamino acid residues 1-23, 35-49, 57-88 and 96-108, respectively, of SEQID NO:2.

In one embodiment, the present invention relates to humanized monoclonalantibodies having binding specificity for GD2, wherein the antibodyhu3F8-H1L1-IgG1 comprises heavy chain variable domain of SEQ ID NO:4 andlight chain variable domain of SEQ ID NO:5, wherein hu3F8H2L2-IgG1comprises heavy chain variable domain SEQ ID NO:6 and light chainvariable domain SEQ ID NO:7, wherein hu3F8-H1L1-IgG4 comprises heavychain variable domain SEQ ID NO:8 and light chain variable domain SEQ IDNO:5 or 7. Such humanized antibodies are derived from the humanizationof the murine 3F8 antibody. In certain embodiments, the humanizedantibodies comprise a heavy chain whose complementarity determiningregions (CDR) 1, 2 and 3 are defined by amino acid residues 31-35, 50-64and 98-108, respectively, of SEQ ID NO:4, 6 or 8. In certainembodiments, the humanized antibodies comprise a light chain whose CDRs1, 2 and 3 are defined by amino acid residues 24-34, 50-56 and 89-95,respectively, of SEQ ID NO:5 or 7.

The present invention also relates to Anti-GD2 humanized antibodiesengineered with modified carbohydrate composition, hu3F8-IgG1n, withincreased effector function.

The present invention also relates to Hu3F8-IgG1-DEL antibody with atriple mutation DEL (S239D/A330L/I332E) in the heavy chain of hu3F8-IgG1or hu3F8-IgG1n. Hu3F8-IgG1-DEL comprises substitutions in the heavychain consisting of S239D, A330L and I332E of SEQ ID NO: 4, 6, or 8.

The present invention also relates to hu3F8H3L3 derived fromcomputational analysis of the crystal structure and adjustment of theamino acid sequence by backmutation and forward mutation to enhancestability of the antibody. The sequence of the stability enhanced huH3heavy chain is disclosed in SEQ ID NO:9, with the huL3 light chain inSEQ ID NO:10. These mutations can also be introduced into a heavy chainsequence alone or in combination with other mutations the the heavychain described herein.

An additional mutation to enhance stability at the VH-VL interface wasadded at position 43 in the light chain resulted in huL3S. Therefore,huL3S comprises a substitution consisting of Ala43Ser of SEQ ID NO:10.When this change to Serine at position 43 is incorporated in the lightchain of hu3F8-IgG1, that light chain is referred to as huLlS.Similarly, huL1S comprises a substitution consisting of Ala43Ser of SEQID NO: 5 or 7.

Computational modeling further showed that substitution of the Gly atposition 54 to Ile in the CDR3 of the heavy chain would change the shapeof the binding pocket and increase the contact with the GD2 ligand.Adding this mutation Gly54Ile to the CDR regions of the sequences ofhuH1-IgG1 and huH3-IgG1 resulted in huH1I-gamma1 and huH3I-gamma,respectively. Therefore, huHI-gamma1 comprises a Gly54Ile substitutionin SEQ ID NO:4, 6, or 8. huH3I-gamma1 comprises a Gly54Ile substitutionin SEQ ID NO:9.

Preferred antibodies of the present invention are those that bind humanGD2 and perform the desired function, i.e. effector function, blockingpain, or inhibiting cell growth. Preferred methods for determiningmonoclonal antibody specificity and affinity by competitive inhibitioncan be found in Harlow, et al, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), herebyincorporated by reference thereto. At least one antibody of theinvention binds at least one specified epitope specific to human GD2,subunit, fragment, portion or any combination thereof. The epitope cancomprise at least one antibody binding region, which epitope ispreferably comprised of at least 1-5 sugar residues or ceramide of atleast one portion of GD2.

In one aspect, the present invention provides at least one isolatedhumanized Anti-GD2 antibody, comprising at least one variable regionfrom m3F8, and the nucleic acid sequences encoding same.

In another aspect, the present invention provides at least one isolatedmammalian Anti-GD2 antibody, comprising either (i) all of the heavychain complementarity determining regions (CDR) amino acid sequencesderived from m3F8, and the nucleic acid sequences encoding them; or (ii)all of the light chain CDR amino acids sequences from m3F8, and thenucleic acid sequences encoding them.

In another aspect, the present invention provides at least one isolatedmammalian Anti-GD2 antibody, comprising at least one heavy chain orlight chain CDR having the amino acid sequence derived from m3F8, andthe nucleic acid sequences encoding them.

In another aspect the present invention provides at least one isolatedchimeric, humanized or CDR-grafted Anti-GD2 antibody, comprising atleast one m3F8 CDR, wherein the antibody specifically binds at least oneepitope comprising at least 1-5 sugar residues or ceramide of an epitopeof human GD2.

In yet another aspect, the present invention provides adiagnostic/detection or therapeutic immunoconjugate comprising anantibody component that comprises any of the 3F8 MoAbs or fragmentsthereof of the present invention, or an antibody fusion protein orfragment thereof that comprises any of the 3F8 antibodies or fragmentsthereof of the present invention, wherein the antibody component isbound to at least one diagnostic/detection agent or at least onetherapeutic agent.

In still another aspect, the present invention provides a therapeuticimmunoconjugate comprising a therapeutic agent that is selected from thegroup consisting of a radionuclide, boron, gadolinium or uranium atoms,an immunomodulator, such as a cytokine, a stem cell growth factor, alymphotoxin, such as tumor necrosis factor (TNF), a hematopoietic factorsuch as an interleukin (IL), a colony stimulating factor (CSF) such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF)), an interferon (IFN) suchas interferons-alpha, -beta or -gamma, and a stem cell growth factor, ahematopoietic factor, erythropoietin, thrombopoietin, an antibody, ahormone, a hormone antagonist, an enzyme, an enzyme inhibitor, aphotoactive therapeutic agent, a cytotoxic drug, such as antimitotic,alkylating, antimetabolite, angiogenesis-inhibiting, apoptotic,alkaloid, COX-2-inhibiting and antibiotic agents, a cytotoxic toxin,such as plant, microbial, and animal toxins, and a synthetic variationsthereof, an angiogenesis inhibitor, a different antibody and acombination thereof.

In another aspect, the present invention also provides a multivalent,multispecific antibody or fragment thereof comprising one or moreantigen-binding sites having affinity toward an antigen recognized bythe 3F8 antibody and one or more hapten binding sites having affinitytowards epitopes or haptens besides GD2. Preferably, the 3F8 antibody orfragment thereof is chimerized or humanized. Also preferred, theantibody or fragment thereof is fully human or chimerized. In oneembodiment, the multivalent, multispecific antibody or fragment thereofcomprises a diagnostic/detection or therapeutic agent.

In yet another aspect, the present invention provides a method ofdelivering a diagnostic/detection agent, a therapeutic agent, or acombination thereof to a target, comprising: (i) administering to asubject a multivalent, multispecific antibody or fragment thereof of thepresent invention; (ii) waiting a sufficient amount of time for anamount of the non-binding protein to clear the subject's blood stream;and (iii) administering to said subject a carrier molecule comprising adiagnostic/detection agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of said antibody. Thediagnostic/detection agent or said therapeutic agent is selected fromthe group comprising isotopes, dyes, chromagens, contrast agents, drugs,toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormoneantagonists, growth factors, radionuclides, metals, liposomes,nanoparticles, RNA or DNA. The second specificity also includeshapten(s) conjugated to any from the group of agents described. Thesehaptens include, but not limited to biotin and its derivatives, DOTA andits derivatives, DTPA and its derivatives, fluorescein and itsderivatives, histamine and its derivatives, Deferoxamine and itsderivatives).

In any of the methods of the present invention, the subject ispreferably a mammal, such as a human or domestic pet.

In another embodiment of the present invention is a method of treatingor identifying diseased tissues in a subject, comprising: (A)administering to said subject a bi-specific antibody or antibodyfragment having at least one arm that specifically binds a diseasedtissue-associated marker and at least one other arm that specificallybinds a targetable conjugate, wherein said diseased tissue-associatedmarker is an antigen recognized by the 3F8 MoAb; (B) optionally,administering to said subject a clearing composition, and allowing saidcomposition to clear non-localized antibodies or antibody fragments fromcirculation; and (C) administering to said subject a first targetableconjugate which comprises a carrier portion which comprises or bears atleast one epitope recognizable by said at least one other arm of saidbi-specific antibody or antibody fragment, and one or more conjugatedtherapeutic or diagnostic agents. Preferably, at least one arm thatspecifically binds a targeted tissue is a human, chimeric or humanizedAnti-GD2 antibody or a fragment of a human, chimeric or humanizedAnti-GD2 antibody. A preferred embodiment is the use of the 3F8 MoAb insuch applications as a chimeric, humanized, or human antibody, asdescribed herein.

In yet another aspect, the present invention provides a method fordetecting or treating tumors expressing an antigen recognized by a 3F8MoAb in a mammal, comprising: (A) administering an effective amount of abispecific antibody or antibody fragment comprising at least one armthat specifically binds a targeted tissue and at least one other armthat specifically binds a targetable conjugate, wherein said one armthat specifically binds a targeted tissue is a 3F8 antibody or fragmentthereof; and (B) administering a targetable conjugate. The targetableconjugate can be selected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2; (xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2; (xix)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2; flurorescein andits derivatives; desferrioxamine and its derivatives.

In another aspect, the present invention provides a method of targetingwherein the method comprises: (A) injecting a subject who is to undergosuch a procedure with a bispecific antibody F(ab)2 or F(ab′)2 fragment,or single-chain Fv fragment, wherein the bispecific antibody or fragmenthas a first antibody binding site which specifically binds to an antigenrecognized by an 3F8 MoAb, and has a second antibody binding site whichspecifically binds to a hapten, and permitting the antibody fragment toaccrete at target sites; (B) optionally clearing non-targeted antibodyfragments using a clearing agent if the bispecific fragment is notlargely cleared from circulation within about 24 hours of injection, andinjecting a hapten-modified dextran, or dendrimers, or polymers, whichquickly remove nontargeted antibody or fragments into the liver fordegradation (C) detecting the presence of the hapten by nuclear imagingor close-range detection of elevated levels of accreted label at thetarget sites using scanners or probes, within hours of the firstinjection, and conducting said procedure, wherein said detection isperformed without the use of a contrast agent or subtraction agent. In apreferred embodiment, the hapten is labeled with a diagnostic/detectionradioisotope, a MRI image-enhancing agent, a fluorescent label or achemiluminescent label. Fluorescent labels can include rhodamine,fluorescein, renographin, fluorescein isothiocyanate, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.Chemiluminescent labels can include luminol, isoluminol, an aromaticacridinium ester, an imidazole, an acridinium salt and an oxalate ester.MRI image-enhancing agents include gadolinium and ferromagneticsubstances. Imaging of antibody-hapten localization detects intact tumorcells that carry GD2, which is critical for tumor staging, measurementof tumor response to treatment, detection of early relapse and tumorsurveillance. Detection of antibody-hapten localization intraoperativelygives precise location of tumor and uncovers occult sites of disease, toallow complete surgical resection as part of a curative therapy forcancer.

Also considered in the present invention is a multivalent, multispecificantibody or fragment thereof comprising comprising one or moreantigen-binding sites having affinity toward an antigen recognized bythe 3F8 antibody and one or more hapten binding sites having affinitytowards epitopes or haptens on cells (lymphocytes, natural killer cells,neutrophils, myeloid cells, stem cells, neuro stem cells, mesenchymalstem cells, leukemia cells, cytotoxic lymphocytes and B-lymphocytes).These bispecific antibodies or fragments can be administered throughvarious routes, including intravenous, intrathecally, and intratumorallyinto mammals including humans to target endogenous cells or exogenouslyinfused cells to sites or tissues or cells that carry the antigen GD2.Alternatively, cells can be armed ex vivo using these bispecificantibodies or fragments before administration into mammals includinghumans.

Also considered in the present invention is the use of sequences of 3F8or fragments there of, to create chimeric surface receptors specific forGD2 using genetic methods, to redirect cells (lymphocytes, naturalkiller cells, neutrophils, myeloid cells, stem cells, neuro stem cells,mesenchymal stem cells, leukemia cells, cytotoxic lymphocytes andB-lymphocytes) to GD2 bearing tissues, organs or tumors, both fordiagnostic and for therapeutic applications.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding the aforementioned specific Anti-GD2 antibodies, comprising atleast one specified sequence, domain, portion or variant thereof. In amore specific aspect, the present invention provides nucleotidesequences encoding antibodies of the present invention wherein, whereinthe Hu3F8-H1L1-IgG1 (also abbreviated as hu3F8-IgG1) light chain isencoded by SEQ ID NO:11 and the Hu3F8-H1L1-IgG1 heavy chain is encodedby SEQ ID NO:12, wherein Hu3F8-H1L1-IgG4 light chain is encoded by SEQID NO:13 and the Hu3F8-H1L1-IgG4 heavy chain is encoded by SEQ ID NO:14,wherein Hu3F8-H1L2-IgG1 light chain L2 is encoded by SEQ ID NO:15 andthe Hu3F8-H2L2-IgG1 heavy chain H2 is encoded by SEQ ID NO:16. The heavyand light chains of hu3F8-H1L1-IgG1 are interchangeable with the heavyand light chains of hu3F8-H2L2-IgG1, forming Anti-GD2 antibodieshu3F8-H1L2-IgG1 and hu3F8-H2L1-IgG1. Further nucleotide sequences areprovided herein wherein ch3F8-IgG1 light chain is encoded by SEQ IDNO:17 and the ch3F8-IgG1 heavy chain is encoded by SEQ ID NO:18, whereinch3F8-IgG4 light chain is encoded by SEQ ID NO:19 and the ch3F8-IgG4heavy chain is encoded by SEQ ID NO:20, and wherein hu3F8H3L3-IgG1 lightchain L3 is encoded by SEQ ID NO:21 and heavy chain H3 is encoded by SEQID NO:22.

The present invention further provides recombinant vectors comprisingsaid Anti-GD2 antibody nucleic acid molecules, host cells containingsuch nucleic acids and/or recombinant vectors, as well as methods ofmaking and/or using such antibody nucleic acids, vectors and/or hostcells. Thus, the invention comprises isolated nucleic acid encoding atleast one isolated mammalian Anti-GD2 antibody or fragment thereof; anisolated nucleic acid vector comprising the isolated nucleic acid,and/or a prokaryotic or eukaryotic host cell comprising the isolatednucleic acid. The host cell can optionally be at least one selected fromCOS-1, COS-7, HEK293, BHK21, CHO, CHO-S, DG44, BSC-1, Hep G2, 653,SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative,immortalized or transformed cell thereof. Also provided is a method forproducing at least one Anti-GD2 antibody, comprising translating theantibody encoding nucleic acid under conditions in vitro, in vivo or insitu, such that the antibody is expressed in detectable or recoverableamounts, including methods that use vectors which allow proteinexpression to be amplified using growth and survival selection under thecontrol of metabolic pathways or enzymes that include but not limited todhfr (dihydrofolate reductase) or GS (glutamine synthase).

The present invention also provides at least one method for expressingat least one aforementioned Anti-GD2 antibody in a host cell, comprisingculturing a host cell as described herein under conditions wherein atleast one Anti-GD2 antibody is expressed in detectable and/orrecoverable amounts.

The present invention also provides at least one composition comprising(a) an isolated Anti-GD2 antibody encoding nucleic acid and/or antibodyas described herein; and (b) a suitable carrier or diluent. The carrieror diluent can optionally be pharmaceutically acceptable, according toknown carriers or diluents. The composition can optionally furthercomprise at least one further compound, protein or composition. In someof these compositions, the chimeric or humanized antibodies areconjugated to a cytotoxic agent (i.e., an agent that impairs theviability and/or the functions of a cell) such as a cytotoxic drug, atoxin or a radionuclide.

The present invention further provides at least one Anti-GD2 antibodymethod or composition, for administering a therapeutically effectiveamount to modulate or treat at least one GD2 related condition in acell, tissue, organ, animal or patient and/or, prior to, subsequent to,or during a related condition, as known in the art and/or as describedherein. Thus, the invention provides a method for diagnosing or treatinga GD2 related condition in a cell, tissue, organ or animal, comprisingcontacting or administering a composition comprising an effective amountof at least one isolated Anti-GD2 antibody or fragment thereof of theinvention with, or to, the cell, tissue, organ or animal. The method canoptionally further comprise using an effective amount of 0.001-50mg/kilogram of an Anti-GD2 antibody of the invention to the cells,tissue, organ or animal. The method can optionally further comprise thecontacting or the administrating by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrathecal, intra-Ommaya, intravitreous,intraocular, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.The method can optionally further comprise administering, prior,concurrently, or after the antibody contacting or administering at leastone composition comprising an effective amount of at least one compoundor protein or cell selected from at least one of a detectable label orreporter, a TNF antagonist, an antirheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anesthetic, a neuromsucula-r blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, antibody orantibody derived conjugates, an immunosuppressive, a growth hormone, ahormone replacement drug, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analog thereof, a cytotoxic or otheranti-cancer agent, an anti-metabolite such as methotrexate, ananti-proliferative agent, a cytokine, interleukin, growth factors, acytokine antagonist, and an anti-TNFα, white cells, T-cells, LAK cells,TIL cells, natural killer (NK) cells, monocytes, NKT cells, engineered Tcells or NK cells or monocytes or granulocytes.

The present invention further provides at least one Anti-GD2 antibodymethod for diagnosing at least one GD2 related condition in a cell,tissue, organ, animal or patient and/or, prior to, subsequent to, orduring a related condition, as known in the art and/or as describedherein.

The present invention also provides at least one composition, deviceand/or method of delivery for diagnosing of at least one Anti-GD2antibody condition, according to the present invention.

Also provided is a composition comprising at least one isolatedhumanized Anti-GD2 antibody and at least one pharmaceutically acceptablecarrier or diluent. The composition can optionally further comprise aneffective amount of at least one compound or protein selected from atleast one of a detectable label or reporter, a cytotoxic or otheranti-cancer agent, an anti-metabolite such as methotrexate, ananti-proliferative agent, a cytokine, or a cytokine antagonist, a TNFantagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NTHE), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical; an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog.

Also provided is a medical device, comprising at least one isolatedmammalian Anti-GD2 antibody of the invention, wherein the device issuitable to contacting or administering the at least one Anti-GD2antibody by at least one mode selected from parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intrathecal,intra-Ommaya, intravitreous, intraocular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

In a further aspect, the disclosure provides a kit comprising at leastone chimeric or humanized Anti-GD2 antibody or fragment of thedisclosure in lyophilized form in a first container, and an optionalsecond container comprising sterile water, sterile buffered water, or atleast one preservative selected from the group consisting of phenol,m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol,magnesium chloride, alkylparaben, benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, mannitol, sucrose,mannose, other sugars, tween 80, or mixtures thereof in an aqueousdiluent. In one aspect, in the kit, the concentration of Anti-GD2antibody or specified portion or variant in the first container isreconstituted to a concentration of about 0.1 mg/ml to about 500 mg/mlwith the contents of the second container. In another aspect, the secondcontainer further comprises an isotonicity agent. In another aspect, thesecond container further comprises a physiologically acceptable buffer.In one aspect, the disclosure provides a method of treating at least oneGD2 characterized condition, comprising administering to a patient inneed thereof a formulation provided in a kit and reconstituted prior toadministration.

Also provided is an article of manufacture for human pharmaceutical ordiagnostic use, comprising packaging material and a container comprisinga solution or a lyophilized form of at least one isolated chimeric orhumanized Anti-GD2 antibody of the present invention. The article ofmanufacture can optionally comprise having the container as a componentof a parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracerebellar,intracerebroventricular, intrathecal, intra-Ommaya, intravitreous,intraocular, intracolic, intracervical, intragastric, intrahepatic,intramyocardial, intraosteal, intrapelvic, intrapericardiac,intraperitoneal, intrapleural, intraprostatic, intrapulmonary,intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial,intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,buccal, sublingual, intranasal, or transdermal delivery device orsystem.

DETAILED DESCRIPTION

In the description that follows, a number of terms used in recombinantDNA and immunology are extensively utilized. In order to provide aclearer and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

The term “antibody” is art-recognized terminology and is intended toinclude molecules or active fragments of molecules that bind to knownantigens. Examples of active fragments of molecules that bind to knownantigens include Fab and F(ab′)₂, fragments. These active fragments canbe derived from an antibody of the present invention by a number oftechniques. For example, purified monoclonal antibodies can be cleavedwith an enzyme, such as pepsin, and subjected to HPLC gel filtration.The appropriate fraction containing Fab fragments can then be collectedand concentrated by membrane filtration and the like. For furtherdescription of general techniques for the isolation of active fragmentsof antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med.23:1011-1019 (1982). The term “antibody” also includes bispecific andchimeric antibodies.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′).sub.2,F(ab).sub.2, Fab′, Fab, Fv, sFv and the like. Regardless of structure,an antibody fragment binds with the same antigen that is recognized bythe intact antibody. For example, an 3F8 monoclonal antibody fragmentbinds with an epitope recognized by 3F8. The term “antibody fragment”also includes any synthetic or genetically engineered protein that actslike an antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”), and minimal recognitionunits consisting of the amino acid residues that mimic the hypervariableregion.

The language “monoclonal antibody” is art-recognized terminology.Monoclonal antibodies are monospecific antibodies that are the samebecause they are made by one type of immune cell that are all clones ofa unique parent cell.

A variety of methods exist in the art for the production of monoclonalantibodies. For example, the monoclonal antibodies may be made byrecombinant DNA methods, such as those described in U.S. Pat. No.4,816,567. In this context, the term “monoclonal antibody” refers to anantibody derived from a single eukaryotic, phage, or prokaryotic clone.The DNA encoding the monoclonal antibodies of the invention can bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies, or suchchains from human, humanized, or other sources). Once isolated, the DNAmay be placed into expression vectors, which are then transformed intohost cells such as NS0 cells, Simian COS cells, Chinese hamster ovary(CHO) cells,yeast cells,algae cells, eggs, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. The DNA also may bemodified, for example, by substituting the coding sequence for humanheavy and light chain constant domains of a desired species in place ofthe homologous human sequences (U.S. Pat. No. 4,816,567; Morrison et al,supra) or by covalently joining to the immunoglobulin coding sequenceall or part of the coding sequence for a non-immunoglobulin polypeptide.Such a non-immunoglobulin polypeptide can be substituted for theconstant domains of an antibody of the invention, or can be substitutedfor the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.

The term “epitope” is art-recognized. It is generally understood bythose of skill in the art to refer to the region of an antigen orantigens that interacts with an antibody. An epitope of a peptide orprotein or sugar antigen can be linear or conformational, or can beformed by contiguous or noncontinguous amino acid and/or sugar sequencesof the antigen. The GD2 molecule, like many carbohydrates, contain manyepitopes. The epitopes or sugars recognized by the antibodies of thepresent invention and conservative substitutions of these sugars whichare still recognized by the antibody, peptide of chemical mimetics ofthe GD2 antigen, and anti-idiotypic antibodies are an embodiment of thepresent invention. These sugars, or mimetic peptides/chemicals, oranti-idiotypic antibodies, offer a convenient method for eluting GD2 toMoAb or MoAb from GD2 on immunoaffinity columns. Further truncation ofthese epitopes may be possible.

A naked antibody is generally an entire antibody which is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector functions, such as complementfixation and ADCC (antibody-dependent cell cytotoxicity), which setmechanisms into action that may result in cell lysis. Naked antibodiesinclude both polyclonal and monoclonal antibodies, as well as certainrecombinant antibodies, such as chimeric, humanized or human antibodies.However, it is possible that the Fc portion is not required fortherapeutic function, rather an antibody exerts its therapeutic effectthrough other mechanisms, such as induction of cell cycle resting andapoptosis. In this case, naked antibodies also include the unconjugatedantibody fragments defined above.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity-determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species, such as acat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domain of the antibodymolecule is derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a subfragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, nucleases, hormones, immunomodulators, chelators, boroncompounds, photoactive agents or dyes and radioisotopes.

A diagnostic agent is a molecule or atom which is administeredconjugated to an antibody moiety, i.e., antibody or antibody fragment,or subfragment, and is useful in diagnosing or detecting a disease bylocating the cells containing the antigen. Useful diagnostic agentsinclude, but are not limited to, radioisotopes, dyes (such as with thebiotin-streptavidin complex), contrast agents, fluorescent compounds ormolecules and enhancing agents (e.g., paramagnetic ions) for magneticresonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI techniqueand the preparation of antibodies conjugated to a MRI enhancing agentand is incorporated in its entirety by reference. Preferably, thediagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents for use in magnetic resonance imaging,and fluorescent compounds. In order to load an antibody component withradioactive metals or paramagnetic ions, it may be necessary to react itwith a reagent having a long tail to which are attached a multiplicityof chelating groups for binding the ions. Such a tail can be a polymersuch as a polylysine, polysaccharide, or other derivatized orderivatizable chain having pendant groups to which can be boundchelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crownethers, bis-thiosemicarbazones, polyoximes, and like groups known to beuseful for this purpose. Chelates are coupled to the antibodies usingstandard chemistries. The chelate is normally linked to the antibody bya group which enables formation of a bond to the molecule with minimalloss of immunoreactivity and minimal aggregation and/or internalcross-linking other, more unusual, methods and reagents for conjugatingchelates to antibodies are disclosed in U.S. Pat. No. 4,824,659 toHawthorne, entitled “Antibody Conjugates,” issued Apr. 25, 1989, thedisclosure of which is incorporated herein in its entirety by reference.Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes forradio-imaging. The same chelates, when complexed with non-radioactivemetals, such as manganese, iron and gadolinium are useful for MRI, whenused along with the antibodies of the invention. Macrocyclic chelatessuch as NOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed bythe invention.

An immunoconjugate is a conjugate of an antibody component with atherapeutic or diagnostic agent. The diagnostic agent can comprise aradioactive or non-radioactive label, a contrast agent (such as formagnetic resonance imaging, computed tomography or ultrasound), and theradioactive label can be a gamma-, beta-, alpha-, Auger electron-, orpositron-emitting isotope.

An immunomodulator is a therapeutic agent as defined in the presentinvention that when present, typically stimulates immune cells toproliferate or become activated in an immune response cascade, such asmacrophages, B-cells, and/or T cells. An example of an immunomodulatoras described herein is a cytokine. As the skilled artisan willunderstand, certain interleukins and interferons are examples ofcytokines that stimulate T cell or other immune cell proliferation.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as an transgenicanimal, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell or cells of thehost cells.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and an antigen or epitope. One specificity would be for, forexample, a B-cell, T-cell, myeloid-, plasma-, or mast-cell antigen orepitope. Another specificity could be to a different antigen on the samecell type, such as CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, orCD22 on B-cells. Multispecific, multivalent antibodies are constructsthat have more than one binding site, and the binding sites are ofdifferent specificity. For example, a bispecific diabody, where onebinding site reacts with one antigen and the other with another antigen.

A bispecific antibody is an antibody that can bind simultaneously to twotargets which are of different structure. Bispecific antibodies (bsAb)and bispecific antibody fragments (bsFab) have at least one arm thatspecifically binds to, for example, GD2 and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering. In one form, the bispecific fusionprotein is divalent, consisting of, for example, a scFv with a singlebinding site for one antigen and a Fab fragment with a single bindingsite for a second antigen. In another form, the bispecific fusionprotein is tetravalent, consisting of, for example, an IgG with twobinding sites for one antigen and two identical scFv for a secondantigen.

Recent methods for producing bispecific MoAbs include engineeredrecombinant MoAbs which have additional cysteine residues so that theycrosslink more strongly than the more common immunoglobulin isotypes.See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bispecific fusion proteins canbe produced using molecular engineering.

Bispecific fusion proteins linking two or more different single-chainantibodies or antibody fragments are produced in similar manner.Recombinant methods can be used to produce a variety of fusion proteins.A flexible linker connects the scFv to the constant region of the heavychain of the 3F8 antibody. Alternatively, the scFv can be connected tothe constant region of the light chain of another humanized antibody.Appropriate linker sequences necessary for the in-frame connection ofthe heavy chain Fd to the scFv are introduced into the V_(L) andV_(kappa) domains through PCR reactions. The DNA fragment encoding thescFv is then ligated into a staging vector containing a DNA sequenceencoding the CH1 domain. The resulting scFv-CH1 construct is excised andligated into a vector containing a DNA sequence encoding the V_(H)region of an 3F8 antibody. The resulting vector can be used to transfectan appropriate host cell, such as a mammalian cell for the expression ofthe bispecific fusion protein.

The 3F8 antibodies and fragments thereof of the present invention canalso be used to prepare functional bispecific single-chain antibodies(bscAb), also called diabodies, and can be produced in mammalian cellsusing recombinant methods. See, e.g., Mack et al., Proc. Natl. Acad.Sci., 92: 7021-7025, 1995, incorporated herein by reference. Forexample, bscAb are produced by joining two single-chain Fv fragments viaa glycine-serine linker using recombinant methods. The V light-chain andV heavy-chain domains of two antibodies of interest are isolated usingstandard PCR methods known in the art. Bispecific single-chainantibodies and bispecific fusion proteins are included within the scopeof the present invention.

The ultimate use of the bispecific diabodies described herein is forpre-targeting GD2 positive cells for subsequent specific delivery ofdiagnostic/detection or therapeutic agents. These diabodies bindselectively to targeted antigens allowing for increased affinity and alonger residence time at the desired location. Moreover, non-antigenbound diabodies are cleared from the body quickly and exposure of normaltissues is minimized. The diagnostic/detection and therapeutic agentscan include isotopes, drugs, toxins, cytokines, hormones, growthfactors, conjugates, radionuclides, and metals. For example, gadoliniummetal is used for magnetic resonance imaging (MRI). Radionuclides arealso available as diagnostic and therapeutic agents, especially those inthe energy range of 60 to 4,000 key.

The targetable construct can be of diverse structure, but is selectednot only to avoid eliciting an immune responses, but also for rapid invivo clearance when used within the bsAb targeting method. Hydrophobicagents are best at eliciting strong immune responses, whereashydrophilic agents are preferred for rapid in vivo clearance; thus, abalance between hydrophobic and hydrophilic needs to be established.This is accomplished, in part, by relying on the use of hydrophilicchelating agents to offset the inherent hydrophobicity of many organicmoieties. Also, subunits of the targetable construct may be chosen whichhave opposite solution properties, for example, peptides, which containamino acids, some of which are hydrophobic and some of which arehydrophilic. Aside from peptides, carbohydrates may be used.

Peptides having as few as two amino-acid residues may be used,preferably two to ten residues, if also coupled to other moieties suchas chelating agents. The linker should be a low molecular weightconjugate, preferably having a molecular weight of less than 50,000daltons, and advantageously less than about 20,000 daltons, 10,000daltons or 5,000 daltons, including the metal ions in the chelates.

The presence of hydrophilic chelate moieties on the linker moietieshelps to ensure rapid in vivo clearance. In addition to hydrophilicity,chelators are chosen for their metal-binding properties, and are changedat will since, at least for those linkers whose bsAb epitope is part ofthe peptide or is a non-chelate chemical hapten, recognition of themetal-chelate complex is no longer an issue.

A chelator such as DTPA, DOTA, TETA, or NOTA or a suitable peptide, towhich a detectable label, such as a fluorescent molecule, or cytotoxicagent, such as a heavy metal or radionuclide, can be conjugated. Forexample, a therapeutically useful immunoconjugate can be obtained byconjugating a photoactive agent or dye to an antibody fusion protein.Fluorescent compositions, such as fluorochrome, and other chromogens, ordyes, such as porphyrins sensitive to visible light, have been used todetect and to treat lesions by directing the suitable light to thelesion. In therapy, this has been termed photoradiation, phototherapy,or photodynamic therapy (Jori et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain 22:430 (1986)). Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. Mew et al.,J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380 (1985); Oseroffet al., Proc. Natl. Acad. Sci. USA 83:8744 (1986); idem., Photochem.Photobiol. 46:83 (1987); Hasan et al., Prog. Clin. Biol. Res. 288:471(1989); Tatsuta et al., Lasers Surg. Med. 9:422 (1989); Pelegrin et al.,Cancer 67:2529 (1991). However, these earlier studies did not includeuse of endoscopic therapy applications, especially with the use ofantibody fragments or subfragments. Thus, the present inventioncontemplates the therapeutic use of immunoconjugates comprisingphotoactive agents or dyes.

The same chelators, when complexed with non-radioactive metals, such asMn, Fe and Gd can be used for MRI, when used along with the bsAbs of theinvention. Macrocyclic chelators such as NOTA(1,4,7-triaza-cyclononane-N,N′, N″-triacetic acid), DOTA, and TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are of usewith a variety of metals and radiometals, most particularly withradionuclides of Ga, Y and Cu, respectively.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence or onset of one or more symptoms ofa disorder in a subject as result of the administration of aprophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder. Afirst prophylactic or therapeutic agent can be administered prior to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second prophylactic or therapeutic agent to asubject with a disorder.

“Effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toantibody dependent cell mediated cytotoxicity (ADCC), antibody dependentcell mediated phagocytosis (ADCP), and complement mediated cytotoxicity(CMC). Effector functions include both those that operate after thebinding of an antigen and those that operate independent of antigenbinding.

“Effector cell” as used herein is meant a cell of the immune system thatexpresses one or more Fc receptors and mediates one or more effectorfunctions. Effector cells include but are not limited to monocytes,macrophages, neutrophils, dendritic cells, eosinophils, mast cells,platelets, large granular lymphocytes, Langerhans' cells, natural killer(NK) cells, T-lymphoctes, B-lymphocytes and may be from any organismincluding but not limited to humans, mice, rats, rabbits, and monkeys.

“Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to Fc-gamma-RIIA (CD32A), Fc-gamma-RIIB (CD32B), Fc-gamma-RIIIA(CD16A), Fc-gamma-RIIIB (CD16B), Fc-gamma-RI (CD64), Fc-epsilon-RII(CD23), FcRn, C1q, C3, staphylococcal protein A, streptococcal proteinG, and viral Fc.gamma.R. Fc ligands may include undiscovered moleculesthat bind Fc.

In a preferred specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule has an altered affinity for an FcγR, providedthat said variant Fc region does not have a substitution at positionsthat make a direct contact with FcγR based on crystallographic andstructural analysis of Fc-FcγR interactions such as those disclosed bySondermann et al., 2000 (Nature, 406: 267-273 which is incorporatedherein by reference in its entirety). Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(hinge region), amino acids 265-269 (B/C loop), amino acids 297-299(C′/E loop), and amino acids 327-332 (F/G) loop. In some embodiments,the molecules of the invention comprising variant Fc regions comprisemodification of at least one residue that makes a direct contact with anFcγR based on structural and crystallographic analysis.

One aspect of the invention includes 3F8 antibody with alteredaffinities for activating and/or inhibitory receptors, having variant Fcregions with one or more amino acid modifications, wherein said one ormore amino acid modification is a substitution at position 239 withaspartic acid, at position 330 with Leucine and at position 332 withglutamic acid (See Example 13).

The invention encompasses molecules comprising a variant Fc region withadditions, deletions, and/or substitutions to one or more amino acid inthe Fc region of an antibody of the present invention in order to altereffector function, or enhance or diminish affinity of antibody to FcR.These mutations are within the skill of a person in the art. Therefore,the invention encompasses molecules comprising variant Fc regions thatbinds with a greater affinity to one or more FcγRs. Such moleculespreferably mediate effector function more effectively as discussedinfra. In other embodiments, the invention encompasses moleculescomprising a variant Fc region that bind with a weaker affinity to oneor more FcγRs. Reduction or elimination of effector function isdesirable in certain cases for example in the case of antibodies whosemechanism of action involves blocking or antagonism but not killing ofthe cells bearing a target antigen. Reduction or elimination of effectorfunction would be desirable in cases of autoimmune disease where onewould block FcγR activating receptors in effector cells (This type offunction would be present in the host cells). In general increasedeffector function would be directed to tumor and foreign cells.

The Fc variants of the present invention may be combined with other Fcmodifications, including but not limited to modifications that altereffector function. The invention encompasses combining an Fc variant ofthe invention with other Fc modifications to provide additive,synergistic, or novel properties in antibodies or Fc fusions. Preferablythe Fc variants of the invention enhance the phenotype of themodification with which they are combined. For example, if an Fc variantof the invention is combined with a mutant known to bind FcγRIIIA with ahigher affinity than a comparable molecule comprising a wild type Fcregion; the combination with a mutant of the invention results in agreater fold enhancement in FcγRIIIA affinity. In some embodiments, theFc variants of the present invention are incorporated into an antibodyor Fc fusion that comprises one or more engineered glycoforms, i.e., acarbohydrate composition that is covalently attached to a moleculecomprising an Fc region, wherein said carbohydrate composition differschemically from that of a parent molecule comprising an Fc region.

The invention encompasses antibodies with modified glycosylation sites,preferably without altering the functionality of the antibody, e.g.,binding activity GD2. As used herein, “glycosylation sites” include anyspecific amino acid sequence in an antibody to which an oligosaccharide(i.e., carbohydrates containing two or more simple sugars linkedtogether) will specifically and covalently attach. Oligosaccharide sidechains are typically linked to the backbone of an antibody via eitherN-or O-linkages. N-linked glycosylation refers to the attachment of anoligosaccharide moiety to the side chain of an asparagine residue.O-linked glycosylation refers to the attachment of an oligosaccharidemoiety to a hydroxyamino acid, e.g., serine, threonine. An Fc-glycoform,hu3F8-H1L1-IgG1n that lacked certain oligosaccharides including fucoseand terminal N-acetylglucosamine was produced in special CHO cells andexhibited enhanced ADCC effector function.

In some embodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by adding ordeleting a glycosylation site. Methods for modifying the carbohydratecontent of antibodies are well known in the art and encompassed withinthe invention, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S.Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No.2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No. 6,472,511; all ofwhich are incorporated herein by reference in their entirety. In otherembodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by deleting one ormore endogenous carbohydrate moieties of the antibody. In a specificembodiment, the invention encompasses deleting the glycosylation site ofthe Fc region of an antibody, by modifying position 297 from asparagineto alanine.

Engineered glycoforms may be useful for a variety of purposes, includingbut not limited to enhancing or reducing effector function. Engineeredglycoforms may be generated by any method known to one skilled in theart, for example by using engineered or variant expression strains, byco-expression with one or more enzymes, for example DIN-acetylglucosaminyltransferase III (GnTI11), by expressing a moleculecomprising an Fc region in various organisms or cell lines from variousorganisms, or by modifying carbohydrate(s) after the molecule comprisingFc region has been expressed. Methods for generating engineeredglycoforms are known in the art, and include but are not limited tothose described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davieset al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473)U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1;PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton,N.J.); GlycoMAb™Mglycosylation engineering technology (GLYCARTbiotechnology A G, Zurich, Switzerland); each of which is incorporatedherein by reference in its entirety. See, e.g., WO 00061739; EA01229125;US 20030115614; Okazaki et al., 2004, J M B, 336: 1239-49 each of whichis incorporated herein by reference in its entirety.

As used herein, the term “derivative” in the context of polypeptides orproteins refers to a polypeptide or protein that comprises an amino acidsequence which has been altered by the introduction of amino acidresidue substitutions, deletions or additions. The term “derivative” asused herein also refers to a polypeptide or protein which has beenmodified, i.e, by the covalent attachment of any type of molecule to thepolypeptide or protein. For example, but not by way of limitation, anantibody may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. A derivative polypeptide or protein may beproduced by chemical modifications using techniques known to those ofskill in the art, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Further, a derivative polypeptide or protein derivative possesses asimilar or identical function as the polypeptide or protein from whichit was derived.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide.

Effective Amount: As used herein, the term “effective amount” refers toan amount of a given compound, conjugate or composition that isnecessary or sufficient to realize a desired biologic effect. Aneffective amount of a given compound, conjugate or composition inaccordance with the methods of the present invention would be the amountthat achieves this selected result, and such an amount can be determinedas a matter of routine by a person skilled in the art, using assays thatare known in the art and/or that are described herein, without the needfor undue experimentation. For example, an effective amount for treatingor preventing cancer metastasis could be that amount necessary toprevent migration and invasion of a tumor cell across the basementmembrane or across an endothelial layer in vivo. The term is alsosynonymous with “sufficient amount.” The effective amount for anyparticular application can vary depending on such factors as thedisease, disorder or condition being treated, the particular compositionbeing administered, the route of administration, the size of thesubject, and/or the severity of the disease or condition. One ofordinary skill in the art can determine empirically the effective amountof a particular compound, conjugate or composition of the presentinvention, in accordance with the guidance provided herein, withoutnecessitating undue experimentation.

As used herein in connection with a measured quantity, the term “about”refers to the normal variation in that measured quantity that would beexpected by the skilled artisan making the measurement and exercising alevel of care commensurate with the objective of the measurement and theprecision of the measuring equipment used. Unless otherwise indicated,“about” refers to a variation of +/−10% of the value provided.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to Anti-GD2 antibodies or antibody polypeptidesinclude any polypeptides which retain at least some of theantigen-binding properties of the corresponding native antibody orpolypeptide, i.e., those polypeptides that retain the ability to bind toone or more epitopes on GD2. Fragments of polypeptides of the presentinvention include proteolytic fragments, as well as deletion fragments,in addition to specific antibody fragments discussed elsewhere herein.Variants of Anti-GD2 antibodies and antibody polypeptides useful inaccordance with the present invention include fragments as describedabove, and also polypeptides with altered amino acid sequences due toamino acid substitutions, deletions, or insertions. Variants may occurnaturally or be non-naturally occurring. Non-naturally occurringvariants may be produced using art-known mutagenesis techniques orunnatural amino aicds. Variant polypeptides may comprise conservative ornon-conservative amino acid substitutions, deletions or additions.Derivatives of Anti-GD2 antibodies and antibody polypeptides useful inaccordance with the present invention are polypeptides which have beenaltered so as to exhibit additional features not found on the nativepolypeptide. Examples include fusion proteins. Variant polypeptides mayalso be referred to herein as “polypeptide analogs.” As used herein a“derivative” of an Anti-GD2 antibody or antibody polypeptide refers to asubject polypeptide having one or more residues chemically derivatizedby reaction of a functional side group. Also included as “derivatives”are those peptides which contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids. For example,4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine.

Treatment: As used herein, the terms “treatment,”“treat,” “treated” or“treating” refer to prophylaxis and/or therapy, particularly wherein theobject is to prevent or slow down (lessen) an undesired physiologicalchange or disorder, such as the progression of multiple sclerosis.Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented. By “subject” or “individual”or “animal” or “patient” or “mammal,” is meant any subject, particularlya mammalian subject, for whom diagnosis, prognosis, or therapy isdesired. Mammalian subjects include humans and other primates, domesticanimals, farm animals, and zoo, sports, or pet animals such as dogs,cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and thelike.

Humanized Antibodies

In one embodiment, the antibodies provided by the present invention aremonoclonal antibodies, which in a preferred embodiment are humanizedversions of cognate Anti-GD2 antibodies derived from other species. Ahumanized antibody is an antibody produced by recombinant DNAtechnology, in which some or all of the amino acids of a humanimmunoglobulin light or heavy chain that are not required for antigenbinding (e.g., the constant regions and the framework regions of thevariable domains) are used to substitute for the corresponding aminoacids from the light or heavy chain of the cognate, nonhuman antibody.By way of example, a humanized version of a murine antibody to a givenantigen has on both of its heavy and light chains (1) constant regionsof a human antibody; (2) framework regions from the variable domains ofa human antibody; and (3) CDRs from the murine antibody. When necessary,one or more residues in the human framework regions can be changed toresidues at the corresponding positions in the murine antibody so as topreserve the binding affinity of the humanized antibody to the antigen.This change is sometimes called “back mutation.” Similarly, forwardmutations may be made to revert back to murine sequence for a desiredreason, e.g. stability or affinity to antigen. For example, forhu3F8-H1L1-IgG1 backmutations were necessary at 19 positions in theheavy chain sequence and 17 positions in the light chain in order tomaintain the in vitro affinity of binding. Humanized antibodiesgenerally are less likely to elicit an immune response in humans ascompared to chimeric human antibodies because the former containconsiderably fewer non-human components.

Suitable methods for making the humanized antibodies of the presentinvention are described in, e.g., Winter EP 0 239 400; Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239: 1534-1536 (1988); Queen et al., Proc.Nat. Acad. ScL USA 86:10029 (1989); U.S. Pat. No. 6,180,370; and Orlandiet al., Proc. Natl. Acad. Sd. USA 86:3833 (1989); the disclosures of allof which are incorporated by reference herein in their entireties.Generally, the transplantation of murine (or other non-human) CDRs ontoa human antibody is achieved as follows. The cDNAs encoding heavy andlight chain variable domains are isolated from a hybridoma. The DNAsequences of the variable domains, including the CDRs, are determined bysequencing. The DNAs, encoding the CDRs are inserted into thecorresponding regions of a human antibody heavy or light chain variabledomain coding sequences, attached to human constant region gene segmentsof a desired isotype (e.g., γl for CH and K for C_(L)), are genesynthesized. The humanized heavy and light chain genes are co-expressedin mammalian host cells (e.g., CHO or NSO cells) to produce solublehumanized antibody. To facilitate large scale production of antibodies,it is often desirable select for high expressor using a DHFR gene or GSgene in the producer line. These producer cell lines are cultured inbioreactors, or hollow fiber culture system, or WAVE technology, toproduce bulk cultures of soluble antibody, or to produce transgenicmammals (e.g., goats, cows, or sheep) that express the antibody in milk(see, e.g., U.S. Pat. No. 5,827,690).

Using the above-described approaches, humanized and chimeric versions ofthe 3F8 antibody, were generated. The cDNAs encoding the murine 3F8variable regions of the light and heavy chains were used to constructvectors for expression of murine-human chimeras in which the murine 3F8variable regions were linked to human IgG1 (for heavy chain) and humankappa (for light chain) constant regions, as described in the Examplesherein. In addition, novel forms of hu3F8 with variant glycosylationwere created, in order to enhance binding to the Fc receptor and enhanceantigen affinity.

In order to produce humanized 3F8 antibodies, the human acceptorframework domains were chosen by homology matching to human germlinesequences. Using these chosen human acceptor frameworks, the light andheavy chain variable domains were designed and a number ofvariants/versions of each were generated and expressed, as describedbelow in Examples.

The nucleotide and amino acid sequence of the heavy and light chainvariable regions of the MoAbs of the invention are described in thisapplication. The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions topolynucleotides that encode an antibody of the present invention.

The polynucleotides may now be obtained by any method known in the art.For example, since the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Since the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, N.Y., which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain or light chain; nucleic acid moleculescomprising the coding sequence for an Anti-GD2 antibody or variableregion; and nucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least one Anti-GD2antibody as described herein and/or as known in the art.

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra).

A vector comprising any of the above-described isolated or purifiednucleic acid molecules, or fragments thereof, is further provided by thepresent invention. Any of the above nucleic acid molecules, or fragmentsthereof, can be cloned into any suitable vector and can be used totransform or transfect any suitable host. The selection of vectors andmethods to construct them are commonly known to persons of ordinaryskill in the art and are described in general technical references (see,in general, “Recombinant DNA Part D,” Methods in Enzymology, Vol. 153,Wu and Grossman, eds., Academic Press (1987)). Desirably, the vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant or animal) into which the vector isto be introduced, as appropriate and taking into consideration whetherthe vector is DNA or RNA. Preferably, the vector comprises regulatorysequences that are specific to the genus of the host. Most preferably,the vector comprises regulatory sequences that are specific to thespecies of the host.

In addition to the replication system and the inserted nucleic acid, theconstruct can include one or more marker genes, which allow forselection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike.

Suitable vectors include those designed for propagation and expansion orfor expression or both. For example, a cloning vector is selected fromthe group consisting of the pUC series, the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI110, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-C1, pMAM and pMAMneo (Clontech). The TOPO cloning system(Invitrogen, Carlsbad, Calif.) also can be used in accordance with themanufacturer's recommendations.

An expression vector can comprise a native or nonnative promoteroperably linked to an isolated or purified nucleic acid molecule asdescribed above. The selection of promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theskill in the art. Similarly, the combining of a nucleic acid molecule,or fragment thereof, as described above with a promoter is also withinthe skill in the art.

Suitable viral vectors include, for example, retroviral vectors,parvovirus-based vectors, e.g., adeno-associated virus (AAV)-basedvectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors,and lentiviral vectors, such as Herpes simplex (HSV)-based vectors.These viral vectors can be prepared using standard recombinant DNAtechniques described in, for example, Sambrook et al., MolecularCloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and John Wiley & Sons,New York, N.Y. (1994).

A retroviral vector is derived from a retrovirus. Retrovirus is an RNAvirus capable of infecting a wide variety of host cells. Upon infection,the retroviral genome integrates into the genome of its host cell and isreplicated along with host cell DNA, thereby constantly producing viralRNA and any nucleic acid sequence incorporated into the retroviralgenome. As such, long-term expression of a therapeutic factor(s) isachievable when using retrovirus. Retroviruses contemplated for use ingene therapy are relatively non-pathogenic, although pathogenicretroviruses exist. When employing pathogenic retroviruses, e.g., humanimmunodeficiency virus (HIV) or human T-cell lymphotrophic viruses(HTLV), care must be taken in altering the viral genome to eliminatetoxicity to the host. A retroviral vector additionally can bemanipulated to render the virus replication-deficient. As such,retroviral vectors are considered particularly useful for stable genetransfer in vivo. Lentiviral vectors, such as HIV-based vectors, areexemplary of retroviral vectors used for gene delivery. Unlike otherretroviruses, HIV-based vectors are known to incorporate their passengergenes into non-dividing cells and, therefore, can be of use in treatingpersistent forms of disease.

Optionally, the isolated or purified nucleic acid molecule, or fragmentthereof, upon linkage with another nucleic acid molecule, can encode afusion protein. The generation of fusion proteins is within the ordinaryskill in the art and can involve the use of restriction enzyme orrecombinational cloning techniques (see, e.g., Gateway™. (Invitrogen)).See, also, U.S. Pat. No. 5,314,995.

In view of the foregoing, the present invention also provides acomposition comprising an above-described isolated or purified nucleicacid molecule, optionally in the form of a vector. The composition cancomprise other components as described further herein.

Also in view of the above, the present invention provides a host cellcomprising an above-described isolated or purified nucleic acidmolecule, optionally in the form of a vector. It is most preferable thatthe cell of the present invention expresses the vector, such that theoligonucleotide, or fragment thereof, is both transcribed and translatedefficiently by the cell. Examples of cells include, but are not limitedto, a human cell, a human cell line, E. coli (e.g., E. coli TB-1, TG-2,DH5α, XL-Blue MRF′ (Stratagene), SA2821 and Y1090), B. subtilis, P.aerugenosa, S. cerevisiae, N. crassa, insect cells (e.g., Sf9, Ea4) andothers set forth herein below. The host cell can be present in a host,which can be an animal, such as a mammal, in particular a human.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules represented in SEQ ID NOS: 1, 2, 4, 7, 8,and the light chain immunoglobulin molecules represented in SEQ ID NOS:2, 5, 6, as well as antibody molecules formed by combinations comprisingthe heavy chain immunoglobulin molecules with light chain immunoglobulinmolecules, such as kappa light chain immunoglobulin molecules, and viceversa, as well as fragments and analogs thereof.

In a specific embodiment, using routine recombinant DNA techniques, oneor more of the CDRs identified herein may be inserted within frameworkregions. The framework regions may be naturally occurring or consensusframework regions, and preferably human framework regions (see, e.g.,Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of humanframework regions). Preferably, the polynucleotide generated by thecombination of the framework regions and CDRs encodes an antibody thatspecifically binds GD2. One or more amino acid substitutions may be madewithin the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/60433, WO 98/24893, WO 98/16664, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of Cole et al., andBoerder et al., are also available for the preparation of humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

Human antibodies produced using other techniques but retaining thevariable regions of the Anti-GD2 antibody of the present invention arepart of this invention. Human antibodies can also be produced usingtransgenic mice which are incapable of expressing functional endogenousmouse immunoglobulins, but which can express human immunoglobulin genes.For example, the human heavy and light chain immunoglobulin genecomplexes may be introduced randomly or by homologous recombination intomouse embryonic stem cells. Alternatively, the human variable region,constant region, and diversity region may be introduced into mouseembryonic stem cells in addition to the human heavy and light chaingenes. The mouse heavy and light chain immunoglobulin genes may berendered non-functional separately or simultaneously with theintroduction of human immunoglobulin loci by homologous recombination.In particular, homozygous deletion of the JH region prevents endogenousantibody production. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, e.g., all or a portion of a polypeptide of theinvention. Monoclonal antibodies directed against the antigen can beobtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol.13:65-93 (1995). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877;U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,886,793; 5,916,771; and 5,939,598, which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose,Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to providehuman antibodies directed against a selected antigen using technologysimilar to that described above.

Also human MoAbs could be made by immunizing mice transplanted withhuman peripheral blood leukocytes, splenocytes or bone marrows (e.g.,Trioma techniques of XTL). Completely human antibodies which recognize aselected epitope can be generated using a technique referred to as“guided selection.” In this approach a selected non-human monoclonalantibody, e.g., a mouse antibody, is used to guide the selection of acompletely human antibody recognizing the same epitope. (Jespers et al.,Bio/technology 12:899-903 (1988)).

As used herein, an “Anti-GD2 antibody”, “Anti-GD2 antibody portion,” or“Anti-GD2 antibody fragment” and/or “Anti-GD2 antibody variant” and thelike include any protein or peptide containing molecule that comprisesat least a portion of an immunoglobulin molecule, containing at leastone complementarity determining region (CDR) of a heavy or light chainor a ligand binding portion thereof derived from a any of the chimericor humanized monoclonal antibodies described herein, in combination witha heavy chain or light chain variable region, a heavy chain or lightchain constant region, a framework region, or any portion thereof, ofnon-murine origin, preferably of human origin, which can be incorporatedinto an antibody of the present invention. Alternatively, the term“Anti-GD2 antibody” shall refer collectively or individually to thechimeric antibody ch3F8-IgG1, ch3F8-IgG4, humanized monoclonalantibodies hu3F8-H1L1-IgG1, hu3F8H1L2-IgG1, hu3F8H2L1-IgG1hu3F8-H2L2-IgG1, hu3F8-H1L1-IgG1n, hu3F8-H1L1-IgG4, hu3F8-H3L3,hu3F8-H1L1S, hu3F8-H3L3S, huH1-I-gamma-1, huH3-I-gamma-1, hu3F8-IgG1-DELantibodies as well as fragments and regions thereof. Such antibody iscapable of modulating, decreasing, antagonizing, mitigating,alleviating, blocking, inhibiting, abrogating and/or interfering with atleast one cell function in vitro, in situ and/or in vivo, wherein saidcell expresses GD2. As a non-limiting example, a suitable Anti-GD2antibody, specified portion or variant of the present invention can bindwith high affinity to an epitope of human GD2.

The term “antibody” is further intended to encompass antibodies,digestion fragments, specified portions and variants thereof, includingantibody mimetics or comprising portions of antibodies that mimic thestructure and/or function of an antibody or specified fragment orportion thereof, including single chain antibodies and fragmentsthereof, each containing at least one CDR derived from an Anti-GD2antibody. Functional fragments include antigen-binding fragments thatbind to a mammalian GD2. For example, antibody fragments capable ofbinding to GD2 or portions thereof, including, but not limited to Fab(e.g., by papain digestion), Fab′ (e.g., by pepsin digestion and partialreduction) and F(ab′)₂ (e.g., by pepsin digestion), facb (e.g., byplasmin digestion), pFc′ (e.g., by pepsin or plasmin digestion), Fd(e.g., by pepsin digestion, partial reduction and reaggregation), Fv orscFv (e.g., by molecular biology techniques) fragments, are encompassedby the invention (see, e.g., Colligan, Immunology, supra).

Antibody fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH₁ domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

As used herein “chimeric” antibodies or “humanized” antibodies or“CDR-grafted” include any combination of the herein described Anti-GD2Abs, or any CDR derived therefrom combined with one or more proteins orpeptides derived from a non-murine, preferably, human antibody. Inaccordance with the invention, chimeric or humanized antibodies includethose wherein the CDR's are derived from one or more of the Anti-GD2 Absdescribed herein and at least a portion, or the remainder of theantibody is derived from one or more human antibodies. Thus, the humanpart of the antibody may include the framework, C_(L), C_(H) domains(e.g., C_(H1), C_(H2), C_(H3)), hinge, (V_(L), V_(H))) regions which aresubstantially non-immunogenic in humans. The regions of the antibodythat are derived from human antibodies need not have 100% identity withhuman antibodies. In a preferred embodiment, as many of the human aminoacid residues as possible are retained in order for the immunogenicityto be negligible, but the human residues may be modified as necessary tosupport the antigen binding site formed by the CDR's whilesimultaneously maximizing the humanization of the antibody. Such changesor variations optionally and preferably retain or reduce theimmunogenicity in humans or other species relative to non-modifiedantibodies. It is pointed out that a humanized antibody can be producedby a non-human animal or prokaryotic or eukaryotic cell that is capableof expressing functionally rearranged human immunoglobulin (e.g., heavychain and/or light chain) genes. Further, when the antibody is a singlechain antibody, it can comprise a linker peptide that is not found innative human antibodies. For example, an Fv can comprise a linkerpeptide, such as two to about twenty glycine or other amino acidresidues, preferably 8-15 glycine or other amino acid residues, whichconnects the variable region of the heavy chain and the variable regionof the light chain. Such linker peptides are considered to be of humanorigin.

Antibody humanization can be performed by, for example, synthesizing acombinatorial library comprising the six CDRs of a non-human targetmonoclonal antibody fused in frame to a pool of individual humanframeworks. A human framework library that contains genes representativeof all known heavy and light chain human germline genes can be utilized.The resulting combinatorial libraries can then be screened for bindingto antigens of interest. This approach can allow for the selection ofthe most favorable combinations of fully human frameworks in terms ofmaintaining the binding activity to the parental antibody. Humanizedantibodies can then be further optimized by a variety of techniques.

Antibody Humanization can be used to evolve mouse or other non-humanantibodies into “fully human” antibodies. The resulting antibodycontains only human sequence and no mouse or non-human antibodysequence, while maintaining similar binding affinity and specificity asthe starting antibody.

For full length antibody molecules, the immunoglobulin genes can beobtained from genomic DNA or mRNA of hybridoma cell lines. Antibodyheavy and light chains are cloned in a mammalian vector system. Assemblyis documented with double strand sequence analysis. The antibodyconstruct can be expressed in other human or mammalian host cell lines.The construct can then be validated by transient transfection assays andWestern blot analysis of the expressed antibody of interest. Stable celllines with the highest productivity can be isolated and screened usingrapid assay methods.

At least one Anti-GD2 antibody of the present invention can beoptionally produced by a cell line, a mixed cell line, an immortalizedcell or clonal population of immortalized cells, as well known in theart. See, e.g., Ausubel, et al., ed., Current Protocols in MolecularBiology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, ColdSpring Harbor, N.Y. (1989); Harlow and Lane, antibodies, a LaboratoryManual, Cold Spring Harbor, N.Y. (1989). Colligan, et al., eds., CurrentProtocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);Colligan et al., Current Protocols in Protein Science, John Wiley &Sons, NY, N.Y., (1997-2001), each entirely incorporated herein byreference.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A), or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart. See, e.g., www.atcc.org, www.lifetech.com., and the like, withantibody producing cells, such as, but not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

Any other suitable host cell can also be used for expressingheterologous or endogenous nucleic acid encoding an antibody, specifiedfragment or variant thereof, of the present invention. The fused cells(hybridomas) or recombinant cells can be isolated using selectiveculture conditions or other suitable known methods, and cloned bylimiting dilution or cell sorting, or other known methods. Cells whichproduce antibodies with the desired specificity can be selected by asuitable assay (e.g., ELISA).

Antibodies of the present invention can also be prepared using at leastone Anti-GD2 antibody encoding nucleic acid to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. Such animals can be providedusing known methods. See, e.g., but not limited to, U.S. Pat. Nos.5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616, 5,565,362;5,304,489, and the like, each of which is entirely incorporated hereinby reference.

Antibodies of the present invention can additionally be prepared usingat least one Anti-GD2 antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited totobacco and maize) that produce such antibodies, specified portions orvariants in the plant parts or in cells cultured therefrom. As anon-limiting example, transgenic tobacco leaves expressing recombinantproteins have been successfully used to provide large amounts ofrecombinant proteins, e.g., using an inducible promoter. See, e.g.,Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.Antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencescited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to known methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October,1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem Soc. Trans.22:940-944 (1994); and references cited therein. Each of the abovereferences is entirely incorporated herein by reference.

An Anti-GD2 antibody can be recovered and purified from recombinant cellcultures by well-known methods including, but not limited to, protein Apurification, protein G purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, eachentirely incorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

Purified antibodies can be characterized by, for example, ELISA,ELISPOT, flow cytometry, immunocytology, fliacore™ analysis, SapidyneKinExA™ kinetic exclusion assay, SDS-PAGE and Western blot, or by HPLCanalysis as well as by a number of other functional assays disclosedherein.

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pIRES1neo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as DHFR, GPT, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

In accordance with the present invention, the Anti-GD2 antibodiescomprise any one of ch3F8-IgG1, ch3F8-IgG4, hu3F8-H1L1-IgG1,hu3F8-H1L2-IgG1, hu3F8-H2L1-IgG1, hu3F8-H2L2-IgG1, hu3F8-H1L1-IgG1n,hu3F8-H1L1-IgG4, hu3F8-H3L3, hu3F8-H1L1S, hu3F8-H3L3S, huH1I-gamma-1,huH3I-gamma-1, hu3F8-IgG1-DEL antibodies or an antibody in which thevariable region or CDRs are derived from any one of ch3F8-IgG1,ch3F8-IgG4, hu3F8-H1L1-IgG1, hu3F8-H1L2-IgG1, hu3F8-H2L1-IgG1,hu3F8-H2L2-IgG1, hu3F8-H1L1-IgG1n, hu3F8-H1L1-IgG4, hu3F8-H3L3,hu3F8-H1L1S, hu3F8-H3L3S, huH1I-gamma1, huH3I-gamma1, hu3F8-IgG1-DELantibody and the framework and constant regions of the antibody arederived from one or more human antibodies. The variable region or CDRsderived from the antibody preferably have from about 90% to about 100%identity with the variable region or CDRs of any one of ch3F8-IgG1,ch3F8-IgG4, hu3F8-H1L1-IgG1, hu3F8-H1L2-IgG1, hu3F8-H2L1-IgG1,hu3F8-H2L2-IgG1, hu3F8-H1L1-IgG1n, hu3F8-H1L1-IgG4, hu3F8-H3L3,hu3F8-H1L1S, hu3F8-H3L3S, huH1I-gamma1, huH3I-gamma1, hu3F8-IgG1-DELalthough any and all modifications, including substitutions, insertionsand deletions, either from natural mutation or from human manipulationare contemplated so long as the antibody maintains the ability to bindto GD2. The regions of the chimeric, humanized or CDR-grafted antibodiesthat are derived from human antibodies need not have 100% identity withthe human antibodies. In a preferred embodiment, as many of the humanamino acid residues as possible are retained in order thatimmunogenicity is negligible, but the human residues, in particularresidues of the framework region, are substituted as required and astaught herein below in accordance with the present invention. Suchmodifications as disclosed herein are necessary to support the antigenbinding site formed by the CDRs while simultaneously maximizing thehumanization of the antibody.

Amino acid sequences that are substantially the same as the sequencesdescribed herein include sequences comprising conservative amino acidsubstitutions, as well as amino acid deletions and/or insertions. Aconservative amino acid substitution refers to the replacement of afirst amino acid by a second amino acid that has chemical and/orphysical properties (e.g., charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include replacement of one aminoacid by another within the following groups: lysine (K), arginine (R)and histidine (H); aspartate (D) and glutamate (E); asparagine (N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D andE; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) andglycine (G); F, W and Y; C, S and T.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given Anti-GD2 antibody, fragment or variant willnot be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, asspecified herein.

Amino acids in an Anti-GD2 antibody of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g.,Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science244:1081-1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least binding to GD2. Sites that are critical for antibodybinding can also be identified by structural analysis such ascrystallization, nuclear magnetic resonance or photoaffinity labeling(Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al.,Science 255:306-312 (1992)).

An Anti-GD2 antibody can further optionally comprise a polypeptide of atleast one of 70-100% of the contiguous amino acids of the CDRs derivedfrom at least one of sequence described herein.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of at least onesequence in Tables 1-4.

Exemplary heavy chain and light chain variable regions sequences areprovided herein. The antibodies of the present invention, or specifiedvariants thereof, can comprise any number of contiguous amino acidresidues from an antibody of the present invention, wherein that numberis selected from the group of integers consisting of from 10-100% of thenumber of contiguous residues in an Anti-GD2 antibody. Optionally, thissubsequence of contiguous amino acids is at least about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250 or more amino acids in length, or any rangeor value therein. Further, the number of such subsequences can be anyinteger selected from the group consisting of from 1 to 20, such as atleast 2, 3, 4, or 5.

In accordance with the present invention, the nucleic acid sequences setforth in SEQ ID NOs: 11-22 and the deduced amino acid sequences of thevariable regions (light and heavy chain) of the Anti-GD2 antibodies areset forth in SEQ ID NOs:1-10. Each of the heavy and light chain variableregions contain three CDRs that combine to form the antigen bindingsite. The three CDRs are surrounded by four framework regions thatprimarily function to support the CDRs. The sequences of the CDRs withinthe sequences of the variable regions of the heavy and light chains canbe identified by computer-assisted alignment according to Kabat et al.(1987) in Sequences of Proteins of Immunological Interest, 4th ed.,United States Department of Health and Human Services, U.S. GovernmentPrinting Office, Washington, D.C., or by molecular modeling of thevariable regions, for example utilizing the ENCAD program as describedby Levitt (1983) J. Mol. Biol. 168:595.

Human genes which encode the constant (C) regions of the humanizedantibodies, fragments and regions of the present invention can bederived from a human fetal liver library, by known methods. Human Cregion genes can be derived from any human cell including those whichexpress and produce human immunoglobulins. The human C_(H) region can bederived from any of the known classes or isotypes of human H chains,including gamma, mu, alpha, delta, epsilon, and subtypes thereof, suchas G1, G2, G3 and G4. Since the H chain isotype is responsible for thevarious effector functions of an antibody, the choice of C_(H) regionwill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity(ADCC). Preferably, the C_(H) region is derived from gamma 1 (IgG1) orgamma 4 (IgG4).

The human C_(L) region can be derived from either human L chain isotype,kappa or lambda, preferably kappa.

Genes encoding human immunoglobulin C regions are obtained from humancells by standard cloning techniques (Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2.sup.nd Edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., eds. CurrentProtocols in Molecular Biology (1987-1993)). Human C region genes arereadily available from known clones containing genes representing thetwo classes of L chains, the five classes of H chains and subclassesthereof.

The sequences of the variable regions of the antibody may be modified byinsertions, substitutions and deletions to the extent that the chimericantibody maintains the ability to bind to human GD2. The ordinarilyskilled artisan can ascertain the maintenance of this activity byperforming the functional assays described hereinbelow. The variableregions can have, for example, from about 50% to about 100% homology tothe variable regions identified below. In a preferred embodiment, thevariable regions of the antibody have from about 80% to about 100%homology to the variable regions identified below. In a more preferredembodiment the variable regions have from about 90% to about 100%homology to the variable regions identified below.

In one specific aspect, preferred Anti-GD2 Mabs of the disclosurecomprise variable light chain regions having 95%, 96%, 97%, 98% or 99%amino acid sequence homology to sequences identified herein and furthercomprise variable heavy chain regions having 95%, 96%, 97%, 98% or 99%amino acid sequence homology to sequences in identified herein.

Preferably, the antibody or antigen-binding fragment of an antibody orspecified portion or variant thereof of the present invention bindshuman GD2 and, thereby partially or substantially neutralizes one GD2protein or fragment and thereby inhibit activities mediated through GD2.As used herein, the term “neutralizing antibody” refers to an antibodythat can inhibit GD2 dependent activity by about 20-120%, preferably byat least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay. Thecapacity of an Anti-GD2 antibody to inhibit a GD2-dependent activity ispreferably assessed by at least one suitable assay, as described hereinand/or as known in the art.

As stated, the invention also relates to antibodies, antigen-bindingfragments, immunoglobulin chains and CDRs comprising amino acids in asequence that is substantially the same as an amino acid sequencedescribed herein. Such Anti-GD2 antibodies can include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation, as specified herein. Preferably, suchantibodies or antigen-binding fragments and antibodies comprising suchchains or CDRs can bind human GD2 with high affinity. [0120] As those ofskill in the art will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-100% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody. Each organic moiety that isbonded to an antibody or antigen-binding fragment of the invention canindependently be a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. As used herein, the term “fatty acid”encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane, e.g.polylysine. Thus, an antibody modified by the covalent attachment ofpolylysine is encompassed by the invention. Hydrophilic polymerssuitable for modifying antibodies of the invention can be linear orbranched and include, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate, n-tetradecanoate,n-octadecanoate, n-eicosanoate, n-docosanoate, n-triacontanoate,n-tetracontanoate, cis-.delta.9-octadecanoate, allcis-.delta.5,8,11,14-eicosatetraenoate, octanedioic acid,tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and thelike. Suitable fatty acid esters include mono-esters of dicarboxylicacids that comprise a linear or branched lower alkyl group. The loweralkyl group can comprise from one to about twelve, preferably one toabout six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hernanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—, —NH—, toname a few. Modifying agents that comprise a linker moiety can beproduced, for example, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

The antibodies of the invention can bind human GD2 with a wide range ofaffinities (K_(D)) as shown below.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters are preferably made withstandardized solutions of antibody and antigen, and a standardizedbuffer, such as the buffer described herein.

Anti-GD2 antibodies useful in the methods and compositions of thepresent invention are characterized by binding to GD2 and preferablyhaving low toxicity. In particular, an antibody, specified fragment orvariant of the invention, where the individual components, such as thevariable region, constant region and framework, individually and/orcollectively, optionally and preferably possess low immunogenicity, isuseful in the present invention. The antibodies that can be used in theinvention are optionally characterized by their ability to treatpatients for extended periods with measurable alleviation of symptomsand low and/or acceptable toxicity. Low or acceptable immunogenicityand/or high affinity, as well as other suitable properties, cancontribute to the therapeutic results achieved. “Low immunogenicity” isdefined herein as raising significant HAHA, HACA or HAMA responses inless than about 75%, or preferably less than about 50% of the patientstreated and/or raising low titres in the patient treated (Elliott etal., Lancet 344:1125-1127 (1994), entirely incorporated herein byreference).

Bispecific, heterospecific, heteroconjugate or similar antibodies canalso be used that are monoclonal, humanized, antibodies that havebinding specificities for at least two different antigens. In thepresent case, one of the binding specificities is for at least one GD2protein, the other one is for any other antigen. Methods for makingbispecific antibodies are known in the art. Traditionally, therecombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different specificities (Milstein and Cuello,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986);Chan and Carter, 2010, Nature Rev. 10, 301-316; Weiner et al., 2010,Nature Rev. 10, 317-327; each entirely incorporated herein by reference.

In certain embodiments, the antibodies, that bind to GD2 can be used inunconjugated form. In other embodiments, the antibodies that bind to GD2can be conjugated, e.g., to a detectable label, a drug, a prodrug or anisotope.

In certain methods of the invention described in more detail below, suchas methods of detecting GD2 expression in cells or tissues as a measureof the metastatic potential of tumor cells, or as a way of identifyingin situ carcinomas (e.g., DCIS or LCIS) in tissues, the Anti-GD2antibodies are conjugated to one or more detectable labels. For suchuses, antibodies may be detectably labeled by covalent or non-covalentattachment of a chromogenic, enzymatic, radioisotopic, isotopic,fluorescent, toxic, chemiluminescent, nuclear magnetic resonancecontrast agent or other label.

Examples of suitable chromogenic labels include diaminobenzidine and4-hydroxyazo-benzene-2-carboxylic acid.

Examples of suitable enzyme labels include malate dehydrogenase,staphylococcal nuclease, Δ-5-steroid isomerase, yeast-alcoholdehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholineesterase.

Examples of suitable radioisotopic labels include 3H, ¹¹¹In, ¹²⁵1, ¹³¹1,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, 67Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³¹I-labeled GD2-binding antibodies by the liver. Inaddition, this radionucleotide has a more favorable gamma emissionenergy for imaging (Perkins et al, Eur. J. Nucl. Med. 70:296-301 (1985);Carasquillo et ah, J. Nucl. Med. 25:281-287 (1987)). For example, ¹¹¹Incoupled to monoclonal antibodies with l-(P-isothiocyanatobenzyl)-DPTAhas shown little uptake in non-tumorous tissues, particularly the liver,and therefore enhances specificity of tumor localization (Esteban etal., J. Nucl. Med. 28:861-870 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, aGreen Fluorescent Protein (GFP) label, an o-phthaldehyde label, and afluorescamine label.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include a luminol label, anisoluminol label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and iron.

Typical techniques for binding the above-described labels to Anti-GD2antibodies, are provided by Kennedy et at., Clin. CMm. Acta 70:1-31(1976), and Schurs et al, Clin. CMm. Acta 81:1-40 (1977). Couplingtechniques mentioned in the latter are the glutaraldehyde method, theperiodate method, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

For use in certain therapeutic approaches of the invention such asablation of residual tumor cells following surgery, or prevention ofmetastasis, the Anti-GD2 antibodies can be conjugated to one or moredrugs, prodrugs or isotopes. Preferred such conjugates comprise one ormore ligands, e.g., one or more antibodies or fragments, derivatives orvariants thereof, that bind to GD2, conjugated to one or more cytotoxicagents; such conjugates are useful in the methods of treatment andprevention of tumor metastasis provided by the invention. According tocertain such embodiments of the invention, the Anti-GD2 antibody, isconjugated to a cytotoxic agent. Cytotoxic, e.g., chemotherapeutic,agents useful in the generation of Anti-GD2 antibody-cytotoxic agentconjugates are well known in the art, and include but are not limited tocisplatin, carboplatin, oxaliplatin, paclitaxel, melphalan, doxorubicin,methotrexate, 5-fluorouracil, etoposide, mechlorethamine,cyclophosphamide, bleomycin, microtubule poisons, and annonaceousacetogenins. Other chemotherapeutic agents suitable' for use inaccordance with this aspect of the invention are well- known and will befamiliar to the ordinarily skilled artisan.

The use of conjugates of one or more Anti-GD2 antibody, and one or moresmall molecule toxins, such as a calicheamicin, a maytansine (U.S. Pat.No. 5,208,020), a trichothene, and CC1065, are also contemplated herein.In one embodiment of the invention, the Anti-GD2 antibody is conjugatedto one or more maytansine molecules (e.g. about 1 to about 10 maytansinemolecules per Anti-GD2 antibody). Maytansine may, for example, beconverted to May-SS-Me which may be reduced to May-SH3 and reacted withmodified Anti-GD2 antibody (Chari et al. Cancer Research 52: 127-131(1992)) to generate a maytansinoid-Anti-GD2 antibody conjugate.

Alternatively, the Anti-GD2 antibody can be conjugated to one or morecalicheamicin molecules. The calicheamicin family of antibiotics arecapable of producing double-stranded DNA breaks at sub- picomolarconcentrations. Structural analogues of calicheamicin which may be used(Hinman et al. Cancer Research 53: 3336-3342 (1993) and Lode et al.Cancer Research 58: 2925-2928 (1998)).

Enzymatically active toxins and fragments thereof which can be used toproduce conjugates with one or more Anti-GD2 antibody, includediphtheria A chain, nonbinding active fragments of diphtheria toxin,exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedin the English language on Oct. 28, 1993, the disclosure of which isincorporated herein by reference in its entirety. Mytansinoids may alsobe conjugated to one or more Anti-GD2 antibody.

The present invention further contemplates Anti-GD2 antibody conjugatedwith a compound with nucleolytic activity {e.g., a ribonuclease or a DNAendonuclease such as a deoxyribonuclease; DNase).

A variety of radioactive isotopes are also available for the productionof radioconjugated Anti-GD2 antibody for use in therapeutic methods ofthe invention. Examples include ²¹¹At, ¹³¹I, ¹²⁵1, ⁹⁰Y, ¹⁸⁶ Re, ¹⁸⁸Re,¹⁵³Sm, ²¹²Bi, ³²P and radioactive isotopes of Lu.

Conjugates of the Anti-GD2 antibody and cytotoxic agents may be madeusing a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-I-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), his-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).¹⁴Carbon-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (Mx-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the Anti-GD2 antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release ofthe cytotoxic drug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide- containinglinker (Chari et al. Cancer Research 52:127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the Anti-GD2 antibody ligandand cytotoxic agent may be made, e.g. by recombinant techniques orpeptide synthesis.

The present invention also provides at least one Anti-GD2 antibodycomposition comprising at least one, at least two, at least three, atleast four, at least five, at least six or more Anti-GD2 antibodiesthereof, as described herein and/or as known in the art that areprovided in a non-naturally occurring composition, mixture or form. Suchcompositions comprise non-naturally occurring compositions comprising atleast one or two full length, C- and/or N-terminally deleted variants,domains, fragments, or specified variants, of the Anti-GD2 antibodyamino acid sequence selected from the group consisting of 70-100% of thecontiguous amino acids of the CDR regions of the antibodies describedherein, or specified fragments, domains or variants thereof. PreferredAnti-GD2 antibody compositions include at least one or two full length,fragments, domains or variants as at least one CDR or LBR containingportions of the Anti-GD2 antibody sequences described herein. Furtherpreferred compositions comprise 40-99% of at least one of 70-100% of aCDR region of an Anti-GD2 Ab described herein. Such compositionpercentages are by weight, volume, concentration, molarity, or molalityas liquid or dry solutions, mixtures, suspension, emulsions or colloids,as known in the art or as described herein.

Anti-GD2 antibody compositions of the present invention can furthercomprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneAnti-GD2 antibody to a cell, tissue, organ, animal or patient in need ofsuch modulation, treatment or therapy, optionally further comprising atleast one selected from at least one TNF antagonist (e.g., but notlimited to a TNF antibody or fragment a soluble TNF receptor orfragment, fusion proteins thereof, or a small molecule TNF antagonist),an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquinesulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, anantiviral, a carbapenem, cephalosporin, a fluoroquinolone, a macrolide,a penicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteriod, an anabolic steroid, a diabetes relatedagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an antidiarrheal, an antitussive, an antiemetic, anantiulcer, a laxative, an anticoagulant, an erythropieitin (e.g.,epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim(GM-CSF, Leukine), an immunization, an immunoglobulin, animmunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), agrowth hormone, a hormone replacement drug, an estrogen receptormodulator, a mydriatic, a cycloplegic, an alkylating agent, anantimetabolite, a mitotic inhibitor, a radiopharmaceutical, anantidepressant, antimanic agent, an antipsychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog, domase alpha(Pulmozyme), a cytokine or a cytokine antagonist, and cell therapies.Non-limiting examples of such cytokines include, but are not limited to,any of IL-1 to IL-34. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference.

Such anti-cancer or anti-infectives can also include toxin moleculesthat are associated, bound, co-formulated or co-administered with atleast one antibody of the present invention. The toxin can optionallyact to selectively kill the pathologic cell or tissue. The pathologiccell can be a cancer or other cell. Such toxins can be, but are notlimited to, purified or recombinant toxin or toxin fragment comprisingat least one functional cytotoxic domain of toxin, e.g., selected fromat least one of ricin, diphtheria toxin, a venom toxin, or a bacterialtoxin. The term toxin also includes both endotoxins and exotoxinsproduced by any naturally occurring, mutant or recombinant bacteria orviruses which may cause any pathological condition in humans and othermammals, including toxin shock, which can result in death. Such toxinsmay include, but are not limited to, enterotoxigenic E. coli heat-labileenterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin,Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-1),Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcalenterotoxins and the like. Such bacteria include, but are not limitedto, strains of a species of enterotoxigenic E. coli (ETEC),enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcuspyogenes), Shigella species (e.g., Shigella dysenteriae, Shigellaflexneri, Shigella boydii, and Shigella sonnei), Salmonella species(e.g., Salmonella typhi, Salmonella cholerasuis, Salmonellaenteritidis), Clostridium species (e.g., Clostridium perfringens.Clostridium dificile, Clostridium botulinum), Camphlobacter species(e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter species,(e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria,Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibriosparahemolyticus), Klebsiella species, Pseudomonas aeruginosa, andStreptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, N.Y. (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

Anti-GD2 antibody compounds, compositions or combinations of the presentinvention can further comprise at least one of any suitable auxiliary,such as, but not limited to, diluent, binder, stabilizer, buffers,salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18.sup.th Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the Anti-GD2 antibody, fragment or variantcomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-GD2 antibody compositions can also include a buffer or a pHadjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts such as citrate.

Additionally, Anti-GD2 antibody compositions of the invention caninclude polymeric excipients/additives such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the Anti-GD2 antibody, portion or variantcompositions according to the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 19.sup.thed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), thedisclosures of which are entirely incorporated herein by reference.Preferred carrier or excipient materials are carbohydrates (e.g.,saccharides and alditols) and buffers (e.g., citrate) or polymericagents.

As noted above, the invention provides for stable formulations, which ispreferably a phosphate buffer with saline or a chosen salt, as well aspreserved solutions and formulations containing a preservative as wellas multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one Anti-GD2 antibody in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001-5%, or any rangeor value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g.,0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9,1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one Anti-GD2 antibody with the prescribed buffersand/or preservatives, optionally in an aqueous diluent, wherein saidpackaging material comprises a label that indicates that such solutioncan be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30,36, 40, 48, 54, 60, 66, 72 hours or greater. The invention furthercomprises an article of manufacture, comprising packaging material, afirst vial comprising lyophilized at least one Anti-GD2 antibody, and asecond vial comprising an aqueous diluent of prescribed buffer orpreservative, wherein said packaging material comprises a label thatinstructs a patient to reconstitute the at least one Anti-GD2 antibodyin the aqueous diluent to form a solution that can be held over a periodof twenty-four hours or greater.

The at least one Anti-GD2 antibody used in accordance with the presentinvention can be produced by recombinant means, including from mammaliancell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

The range of at least one Anti-GD2 antibody in the product of thepresent invention includes amounts yielding upon reconstitution, if in awet/dry system, concentrations from about 1.0 microgram/ml to about 1000mg/ml, although lower and higher concentrations are operable and aredependent on the intended delivery vehicle, e.g., solution formulationswill differ from transdermal patch, pulmonary, transmucosal, or osmoticor micro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one Anti-GD2 antibody and a preservativeselected from the group consisting of phenol, m-cresol, p-cresol,o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one Anti-GD2 antibody andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one Anti-GD2 antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least one Anti-GD2antibody that is reconstituted with a second vial containing water, apreservative and/or excipients, preferably a phosphate buffer and/orsaline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2° C. to about 40° C. and retain the biologically activity of theprotein for extended periods of time, thus, allowing a package labelindicating that the solution can be held and/or used over a period of 6,12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent isused, such label can include use up to 1-12 months, one-half, one and ahalf, and/or two years.

The solutions of at least one Anti-GD2 antibody in the invention can beprepared by a process that comprises mixing at least one antibody in anaqueous diluent. Mixing is carried out using conventional dissolutionand mixing procedures. To prepare a suitable diluent, for example, ameasured amount of at least one antibody in water or buffer is combinedin quantities sufficient to provide the protein and optionally apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one Anti-GD2antibody that is reconstituted with a second vial containing the aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneAnti-GD2 antibody that is reconstituted with a second vial containingthe aqueous diluent. The clear solution in this case can be up to oneliter or even larger in size, providing a large reservoir from whichsmaller portions of the at least one antibody solution can be retrievedone or multiple times for transfer into smaller vials and provided bythe pharmacy or clinic to their customers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as BD Pens, BDAutojector®, Humaject®, e.g., as made or developed by Becton Dickensen(Franklin Lakes, N.J.,), Disetronic (Burgdorf, Switzerland,; Bioject,Portland, Oreg.; National Medical Products, Weston Medical(Peterborough, UK), Medi-Ject Corp (Minneapolis, Minn.). Recognizeddevices comprising a dual vial system include those pen-injector systemsfor reconstituting a lyophilized drug in a cartridge for delivery of thereconstituted solution such as the HumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one Anti-GD2 antibody in theaqueous diluent to form a solution and to use the solution over a periodof 2-24 hours or greater for the two vial, wet/dry, product. For thesingle vial, solution product, the label indicates that such solutioncan be used over a period of 2-24 hours or greater. The presentlyclaimed products are useful for human pharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one Anti-GD2 antibody and a selectedbuffer, preferably a phosphate buffer containing saline or a chosensalt. Mixing the at least one antibody and buffer in an aqueous diluentis carried out using conventional dissolution and mixing procedures. Toprepare a suitable formulation, for example, a measured amount of atleast one antibody in water or buffer is combined with the desiredbuffering agent in water in quantities sufficient to provide the proteinand buffer at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized atleast one Anti-GD2 antibody that is reconstituted with a second vialcontaining a preservative or buffer and excipients in an aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

At least one Anti-GD2 antibody in either the stable or presentedformulations or solutions described herein, can be administered to apatient in accordance with the present invention via a variety ofdelivery methods including SC or IM injection; transdermal, pulmonary,transmucosal, implant, osmotic pump, cartridge, micro pump, or othermeans appreciated by the skilled artisan, as well-known in the art.

In one embodiment of the present invention, the pharmaceuticalcompositions comprising an Anti-GD2 antibody of the disclosurefacilitate administration of humanized antibodies to an organism,preferably an animal, preferably a mammal. Particular mammals includebovine, canine, equine, feline, ovine, and porcine animals, non-humanprimates, and humans. Humans are particularly preferred.

A high affinity, neutralizing chimeric or human antibody to GD2 would bedesirable to be used in diseases where GD2 is expressed, for example,GD2 is expressed in >50% of melanoma (Zhang et al., 1997, Int. J.Cancer. 73, 42-49), 88% of osteosarcoma (Heiner et al., 1987, CancerRes. 47, 5377-5388), and 93% of soft tissue sarcomas includingliposarcoma, fibrosarcoma, malignant fibrous histiocytoma,leiomyosarcoma, and spindle cell sarcoma (Chang et al., 1992, Cancer 70,633-638), as well as brain tumors (Longee et al., 1991, ActaNeuropathol. 82, 45-54). Anti-GD2 antibodies have been tested inpatients with melanoma (Saleh et al, 1992, Hum. Antibodies Hybridomas 3,19-24; Cheung et al., 1987, J. Clin. Oncol. 5, 1430-1440; Choi et al.,2006, Cancer Immunol. Immunother. 55, 761-774), sarcomas (Choi et al.,2006, supra; Yeh et al., 1992, The fifth Asia and Oceania Congress ofNuclear Medicine and Biology Proceedings, p. 104), small cell lungcancer (Grant et al., 1996, Eur. J. Nucl. Med. 23, 145-149), braintumors (Arbit et al., 1995, Eur. J. Nucl. Med. 22, 419-426), by ivinjection as well as by compartmental therapy using Ommaya reservoirs(Kramer et al., 2007, J. Clin. Oncol. 25, 5465-5470). GD2 is also atumor target for retinoblastoma (Chantada et al., 2006, J. Pediatr.Hematol. Oncol. 28, 369-373) and HTLV-1 infected T cells leukemia cells(Furukawa et al., 1993, PNAS USA 90, 1972-1976). In one preferredaspect, an Anti-GD2 antibody of the disclosure can be used to treatneuroblastoma. Anti-GD2 antibodies or derivatives thereof can be usedeither as a single agent or in combination with other therapeuticagents. In addition, these Mabs can be used as a chemosensitizer wherebytheir use can increase therapeutic efficacy of cytotoxic agents. Theseantibodies can be used as a radiosensitizer whereby their use canimprove efficacy of radiation. They can also be used in combination withother tumor-immunomodulating agents such as IL-2, IL-12 and/or IFNalpha.Additionally, the Anti-GD2 antibodies can be used in combination withother monoclonal antibodies such as anti-TNF-alpha, IL-12/IL-23, IL-2,GpIIb/IIIa receptor, CD52, CD20, RSV proteins, HER2/neu receptor, andthe like; as well as with commercially approved antibodies includingRituxan, Herceptin, Mylotarg, Campath, Zevalin, Bexxar, Erbitux, Avastinand Vectibix.

Thus, the present invention also provides a method for modulating ortreating at least one GD2 related disease, in a cell, tissue, organ,animal, or patient, as known in the art or as described herein, using atleast one Anti-GD2 antibody of the present invention.

The present invention includes a method for modulating or treating atleast one malignant disease in a cell, tissue, organ, animal or patient,including, but not limited to, at least one of: multiple myeloma,leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell,T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocyticleukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,myelodysplastic syndrome (MDS), a lymphoma, Hodgkin's disease, amalignant lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, multiplemyeloma, Kaposi's sarcoma, colorectal carcinoma, renal cell carcinoma,pancreatic carcinoma, prostatic carcinoma, nasopharyngeal carcinoma,malignant histiocytosis, paraneoplastic syndrome/hypercalcemia ofmalignancy, solid tumors, adenocarcinomas, sarcomas, malignant melanoma,hemangioma, metastatic disease, cancer related bone resorption, cancerrelated bone pain; the suppression of cancer metastasis; theamelioration of cancer cachexia; and the treatment of inflammatorydiseases such as mesangial proliferative glomerulonephritis and thelike. Such a method can optionally be used in combination with, byadministering before, concurrently or after administration of such GD2antibody, radiation therapy, an anti-angiogenic agent, achemotherapeutic agent, a farnesyl transferase inhibitor or the like.

The present invention also provides a method for modulating or treatingat least one GD2 mediated immune related disease, in a cell, tissue,organ, animal, or patient including, but not limited to, at least one ofrheumatoid arthritis, juvenile rheumatoid arthritis, systemic onsetjuvenile rheumatoid arthritis, psoriatic arthritis, ankylosingspondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis,inflammatory bowel disease, ulcerative colitis, systemic lupuserythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/opticneuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitivity reactions, allergic rhinitis, hayfever, perennial rhinitis, conjunctivitis, endometriosis, asthma,urticaria, systemic anaphylaxis, dermatitis, pernicious anemia,hemolytic disease, thrombocytopenia, graft rejection of any organ ortissue, kidney transplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, hone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, sleep apnea, obesity, heartfailure, sinusitis, inflammatory bowel disease, and the like. See, e.g.,the Merck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J.(1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells etal., eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998,2000), each entirely incorporated by reference.

The present invention also provides a method for modulating or treatingat least one infectious disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: acute orchronic bacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, E. coli0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, vital-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like;

Any of such methods can optionally comprise administering an effectiveamount of at least one composition or pharmaceutical compositioncomprising at least one Anti-GD2 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy.

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one Anti-GD2 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy. Sucha method can optionally further comprise co-administration orcombination therapy for treating such immune diseases or malignantdiseases, wherein the administering of said at least one Anti-GD2antibody, specified portion or variant thereof, further comprisesadministering, before concurrently, and/or after, at least one selectedfrom at least one TNF antagonist (e.g., but not limited to a TNFantibody or fragment, a soluble TNF receptor or fragment, fusionproteins thereof, or a small molecule TNF antagonist), an IL-18 antibodyor fragment, small molecule IL-18 antagonist or IL-18 receptor bindingprotein, an IL-1 antibody (including both IL-1 alpha and IL-1 beta) orfragment, a soluble IL-1 receptor antagonist, an antirheumatic (e.g.,methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalazine, radiation therapy, an anti-angiogenic agent, achemotherapeutic agent, Thalidomidea muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, anantiviral, a carbapenem, cephalosporin, a fluoroquinolone, a macrolide,a penicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteriod, an anabolic steroid, a diabetes relatedagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an erythropieitin (e.g., epoetin alpha), a filgrastim(e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which is entirely incorporated herein byreference.

TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one anti body, specified portion andvariant thereof, of the present invention), include, but are not limitedto, anti-TNF antibodies, antigen-binding fragments thereof, and receptormolecules which bind specifically to TNF; compounds which prevent and/orinhibit TNF synthesis, TNF release or its action on target cells, suchas thalidomide, tenidap, phosphodiesterase inhibitors (e.g.,pentoxifylline and rolipram), A2b adenosine receptor agonists and A2badenosine receptor enhancers; compounds which prevent and/or inhibit TNFreceptor signalling, such as mitogen activated protein (MAP) kinaseinhibitors; compounds which block and/or inhibit membrane TNF cleavage,such as metalloproteinase inhibitors; compounds which block and/orinhibit TNF activity, such as angiotensin converting enzyme (ACE)inhibitors (e.g., captopril); and compounds which block and/or inhibitTNF production and/or synthesis, such as MAP kinase inhibitors.

Any method of the present invention can comprise a method for treating aGD2 mediated disorder or a disorder characterized by GD2 expression,comprising administering an effective amount of a composition orpharmaceutical composition comprising at least one Anti-GD2 antibody toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy. Such a method can optionally further compriseco-administration or combination therapy for treating such immunediseases, wherein the administering of said at least one Anti-GD2antibody, specified portion or variant thereof, further comprisesadministering, before concurrently, and/or after, at least one agent asdescribed above.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one Anti-GD2antibody composition that total, on average, a range from at least about0.01 to 500 milligrams of at least one Anti-GD2 antibody per kilogram ofpatient per dose, and preferably from at least about 0.1 to 100milligrams antibody/kilogram of patient per single or multipleadministration, depending upon the specific activity of contained in thecomposition. Alternatively, the effective serum concentration cancomprise 0.1-5000 ug/ml serum concentration per single or multipleadministration. Suitable dosages are known to medical practitioners andwill, of course, depend upon the particular disease state, specificactivity of the composition being administered, and the particularpatient undergoing treatment in some instances, to achieve the desiredtherapeutic amount, it can be necessary to provide for repeatedadministration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5,5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10,10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5,15, 15.5, 15.9, 1.6, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19,19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 .mu.g/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10milligrams per kilogram per administration or in sustained release formis effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or alternatively or additionally, at least one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20years, or any combination thereof, using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion or lyophilized powder in association, orseparately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1-10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as aqueous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthetic mono- or di- or tri-glycerides. Parental administration isknown in the art and includes, but is not limited to, conventional meansof injections, a gas pressured needle-less injection device as describedin U.S. Pat. No. 5,851,198, and a laser perforator device as describedin U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.

The invention further relates to the administration of at least oneAnti-GD2 antibody by parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intrathecal, intra-Ommaya,intraocular, intravitreous, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.At least one Anti-GD2 antibody composition can be prepared for use forparenteral (subcutaneous, intramuscular or intravenous) or any otheradministration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms such as, but not limited to, creams and suppositories;for buccal, or sublingual administration such as, but not limited to, inthe form of tablets or capsules; or intranasally such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or transdermally such as not limited to a gel, ointment, lotion,suspension or patch delivery system with chemical enhancers such asdimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement”; Hsieh, D. S., Eds., pp. 59-90, MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathwayssuch as electroporation, or to increase the mobility of charged drugsthrough the skin such as iontophoresis, or application of ultrasoundsuch as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

For pulmonary administration, preferably at least one Anti-GD2 antibodycomposition is delivered in a particle size effective for reaching thelower airways of the lung or sinuses. According to the invention, atleast one Anti-GD2 antibody can be delivered by any of a variety ofinhalation or nasal devices known in the art for administration of atherapeutic agent by inhalation. These devices capable of depositingaerosolized formulations in the sinus cavity or alveoli of a patientinclude metered dose inhalers, nebulizers, dry powder generators,sprayers, and the like. Other devices suitable for directing thepulmonary or nasal administration of antibodies are also known in theart. All such devices can use of formulations suitable for theadministration for the dispensing of antibody in an aerosol. Suchaerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles. Metered dose inhalers like the Ventolin®metered dose inhaler, typically use a propellent gas and requireactuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Drypowder inhalers like Turbuhaler™ (Astra), Rotahaler® (Glaxo),Diskus®(Glaxo), devices marketed by Inhale Therapeutics, to name a few,use breath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra,EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No.5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein byreference). Nebulizers like the Ultravent® nebulizer (Mallinckrodt), andthe Acorn II® nebulizer (Marquest Medical Products) (U.S. Pat. No.5,404,871 Aradigm, WO 97/22376), the above references entirelyincorporated herein by reference, produce aerosols from solutions, whilemetered dose inhalers, dry powder inhalers, etc. generate small particleaerosols. These specific examples of commercially available inhalationdevices are intended to be a representative of specific devices suitablefor the practice of this invention, and are not intended as limiting thescope of the invention. Preferably, a composition comprising at leastone Anti-GD2 antibody is delivered by a dry powder inhaler or a sprayer.There are several desirable features of an inhalation device foradministering at least one antibody of the present invention. Forexample, delivery by the inhalation device is advantageously reliable,reproducible, and accurate. The inhalation device can optionally deliversmall dry particles, e.g. less than about 10 um, preferably about 1-5um, for good respirability.

A spray including GD2 antibody composition protein can be produced byforcing a suspension or solution of at least one Anti-GD2 antibodythrough a nozzle under pressure. The nozzle size and configuration, theapplied pressure, and the liquid feed rate can be chosen to achieve thedesired output and particle size. An electrospray can be produced, forexample, by an electric field in connection with a capillary or nozzlefeed. Advantageously, particles of at least one Anti-GD2 antibodycomposition protein delivered by a sprayer have a particle size lessthan about 10 um, preferably in the range of about 1 um to about 5 um,and most preferably about 2 um to about 3 um.

Formulations of at least one Anti-GD2 antibody composition proteinsuitable for use with a sprayer typically include antibody compositionprotein in an aqueous solution at a concentration of about 0.1 mg toabout 100 mg of at least one Anti-GD2 antibody composition protein perml of solution or mg/gm, or any range or value therein, e.g., but notlimited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml ormg/gm. The formulation can include agents such as an excipient, abuffer, an isotonicity agent, a preservative, a surfactant, and,preferably, zinc. The formulation can also include an excipient or agentfor stabilization of the antibody composition protein, such as a buffer,a reducing agent, a bulk protein, or a carbohydrate. Bulk proteinsuseful in formulating antibody composition proteins include albumin,protamine, or the like. Typical carbohydrates useful in formulatingantibody composition proteins include sucrose, mannitol, lactose,trehalose, glucose, or the like. The antibody composition proteinformulation can also include a surfactant, which can reduce or preventsurface-induced aggregation of the antibody composition protein causedby atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxy ethylene sorbitol fatty acidesters. Amounts will generally range between 0.001 and 14% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan monooleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as GD2 antibodies, or specified portions,or variants, can also be included in the formulation.

Antibody composition protein can be administered by a nebulizer, such asjet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer,a compressed air source is used to create a high-velocity air jetthrough an orifice. As the gas expands beyond the nozzle, a low-pressureregion is created, which draws a solution of antibody compositionprotein through a capillary tube connected to a liquid reservoir. Theliquid stream from the capillary tube is sheared into unstable filamentsand droplets as it exits the tube, creating the aerosol. A range ofconfigurations, flow rates, and baffle types can be employed to achievethe desired performance characteristics from a given jet nebulizer. Inan ultrasonic nebulizer, high-frequency electrical energy is used tocreate vibrational, mechanical energy, typically employing apiezoelectric transducer. This energy is transmitted to the formulationof antibody composition protein either directly or through a couplingfluid, creating an aerosol including the antibody composition protein.Advantageously, particles of antibody composition protein delivered by anebulizer have a particle size less than about 10 um, preferably in therange of about 1 um to about 5 um, and most preferably about 2 um toabout 3 um.

Formulations of at least one Anti-GD2 antibody suitable for use with anebulizer, either jet or ultrasonic, typically include a concentrationof about 0.1 mg to about 100 mg of at least one Anti-GD2 antibodyprotein per ml of solution. The formulation can include agents such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one Anti-GD2antibody composition protein, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating at leastone Anti-GD2 antibody composition proteins include albumin, protamine,or the like. Typical carbohydrates useful in formulating at least oneAnti-GD2 antibody include sucrose, mannitol, lactose, trehalose,glucose, or the like. The at least one Anti-GD2 antibody formulation canalso include a surfactant, which can reduce or prevent surface-inducedaggregation of the at least one Anti-GD2 antibody caused by atomizationof the solution in forming an aerosol. Various conventional surfactantscan be employed, such as polyoxyethylene fatty acid esters and alcohols,and polyoxyethylene sorbital fatty acid esters. Amounts will generallyrange between 0.001 and 4% by weight of the formulation. Especiallypreferred surfactants for purposes of this invention are polyoxyethylenesorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like.Additional agents known in the art for formulation of a protein such asantibody protein can also be included in the formulation.

In a metered dose inhaler (MDI), a propellant, at least one Anti-GD2antibody, and any excipients or other additives are contained in acanister as a mixture including a liquefied compressed gas. Actuation ofthe metering valve releases die mixture as an aerosol, preferablycontaining particles in the size range of less than about 10 um,preferably about 1 um to about 5 um, and most preferably about 2 um toabout 3 um. The desired aerosol particle size can be obtained byemploying a formulation of antibody composition protein produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

Formulations of at least one Anti-GD2 antibody for use with ametered-dose inhaler device will generally include a finely dividedpowder containing at least one Anti-IL-6 antibody as a suspension in anon-aqueous medium, for example, suspended in a propellant with the aidof a surfactant. The propellant can be any conventional materialemployed for this purpose, such as chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one Anti-GD2 antibody as a suspensionin the propellant, to protect the active agent against chemicaldegradation, and the like. Suitable surfactants include sorbitantrioleate, soya lecithin, oleic acid, or the like. In some casessolution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein can alsobe included in the formulation.

One of ordinary skill in the art will recognize that the methods of thecurrent invention can be achieved by pulmonary administration of atleast one Anti-GD2 antibody compositions via devices not describedherein.

Formulations for oral administration rely on the co-administration ofadjuvants (e.g., resorcinols and nonionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) toincrease artificially the permeability of the intestinal walls, as wellas the co-administration of enzymatic inhibitors (e.g., pancreatictrypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) toinhibit enzymatic degradation. The active constituent compound of thesolid-type dosage form for oral administration can be mixed with atleast one additive, including sucrose, lactose, cellulose, mannitol,trehalose, raffinose, maltitol, dextran, starches, agar, arginates,chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin,collagen, casein, albumin, synthetic or semisynthetic polymer, andglyceride. These dosage forms can also contain other type(s) ofadditives, e.g., inactive diluting agent, lubricant such as magnesiumstearate, paraben, preserving agent such as sorbic acid, ascorbic acid,alpha.-tocopherol, antioxidant such as cysteine, disintegrator, binder,thickener, buffering agent, sweetening agent, flavoring agent, perfumingagent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug deliver systems for insulin and heparin (U.S. Pat. No.4,239,754). More recently, microspheres of artificial polymers of mixedamino acids (proteinoids) have been used to deliver pharmaceuticals(U.S. Pat. No. 4,925,673). Furthermore, carrier compounds described inU.S. Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used todeliver biologically active agents orally are known in the art.

For absorption through mucosal surfaces, compositions and methods ofadministering at least one Anti-GD2 antibody include an emulsioncomprising a plurality of submicron particles, a mucoadhesivemacromolecule, a bioactive peptide, and an aqueous continuous phase,which promotes absorption through mucosal surfaces by achievingmucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucoussurfaces suitable for application of the emulsions of the presentinvention can include corneal, conjunctival, buccal, sublingual, nasal,vaginal, pulmonary, stomachic, intestinal, and rectal routes ofadministration. Formulations for vaginal or rectal administration, e.g.suppositories, can contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor intranasal administration can be solid and contain as excipients,for example, lactose or can be aqueous or oily solutions of nasal drops.For buccal administration excipients include sugars, calcium stearate,magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No.5,849,695).

For transdermal administration, the at least one Anti-GD2 antibody isencapsulated in a delivery device such as a liposome or polymericnanoparticles, microparticle, microcapsule, or microspheres (referred tocollectively as microparticles unless otherwise stated). A number ofsuitable devices are known, including microparticles made of syntheticpolymers such as polyhydroxy acids such as polylactic acid, polyglycolicacid and copolymers thereof, polyorthoesters, polyanhydrides, andpolyphosphazenes, and natural polymers such as collagen, polyaminoacids, albumin and other proteins, alginate and other polysaccharides,and combinations thereof (U.S. Pat. No. 5,814,599).

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year or more from a singleadministration. Various slow release, depot or implant dosage forms canbe utilized. For example, a dosage form can contain a pharmaceuticallyacceptable non-toxic salt of the compounds that has a low degree ofsolubility in body fluids, for example, (a) an acid addition salt with apolybasic acid such as phosphoric acid, sulfuric acid, citric acid,tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamicacid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, andthe like; (b) a salt with a polyvalent metal cation such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium and the like, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

The following MATERIALS AND METHODS were used in the examples thatfollow.

Materials and Methods

Cell Culture and Human Tissues Human neuroblastoma cell line LAN-1 wasprovided by Dr. Robert Seeger (Children's Hospital of Los Angeles, LosAngeles, Calif.), and NB1691 by Dr. Peter Houghton (St. Jude Children'sResearch Hospital, Memphis, Tenn.). NK-92MI was obtained from AmericanType Culture Collection (ATCC), Manassas, Va. All cell lines were grownin F10 [RPMI 1640 medium supplemented with 10% fetal bovine serum(Hyclone, South Logan, Utah), 2 mM glutamine, 100 U/ml penicillin, and100 ug/ml streptomycin] at 37° C. in a 5% CO₂ incubator. Normal tissuesas well as solid tumor samples of different histological types obtainedat Memorial Sloan-Kettering Cancer Center (MSKCC) were snap frozen inliquid nitrogen. Written informed consent was obtained from the patientsand/or their guardians in accordance to the guidelines of theinstitutional review board of MSKCC.

Monoclonal Antibodies Murine 3F8 was a mouse IgG3 antibody with kappalight chain (Cheung et al., 1985, Cancer Res 45, 2642-9). Monoclonalantibodies 3F8 (mouse IgG3, kappa), 5F11 (mouse IgM, kappa), and 8H9(mouse IgG1, kappa) reactive with neuroblastoma have been previouslydescribed (Cheung et al, 1985, supra; Cheung et al., 2004, J Nucl Med45, 867-77; Modak et al., 2001, Cancer Res 61, 4048-54). They wereproduced as ascites and purified by affinity chromatography: protein A(GE Healthcare, Piscataway, N.J.) for 3F8, protein G for 8H9, andC1q-sepharose (Pierce, Rockford, Ill.) for 5F11. These antibodieswere >90% pure by SDS-PAGE. F(ab′)2 fragments were prepared by pepsindigestion as previously reported (Cheung et al., 1988, J Clin Invest 81,1122-28). Anti-GD2 hybridoma ME361 and TIB114 (N.S.7), a hybridomasecreting an IgG3 control antibody, were obtained from ATCC. 14.G2a waspurchased from BD Biosciences, San Jose, Calif. Chimeric 14.18 waskindly provided by Dr. Stephen Gillies of Lexigen Pharmaceuticals,Lexington, MA. MAB1027 (anti-B7-H3 MoAb) was purchased from R&D System,Minneapolis, Minn. Mouse IgG3 antibody S220-51 specific for GD2 waspurchased from Northstar Bioproducts, Cape Cod, Mass.

Construction of the hu3F8-IgG1, hu3F8-IgG4, ch3F8-IgG1, and ch3F8-IgG4antibody producer lines. Based on human homologs of m3F8, CDR sequencesof both heavy and light chains of m3F8 were grafted into the human IgG1framework and optimized. From two heavy chain and two light chain genes,four versions of hu3F8 were designed. These hu3F8 genes were synthesizedand optimized for CHO cells (Blue Heron Biotechnology, Bothhell, Wash.or Genscript, Piscataway, N.Y.). Using the bluescript vector (Eureka,Calif.), these heavy and light chain genes of hu3F8 were transfectedinto DG44 cells and selected with G418 (InVitrogen, Calif.). Whentransfected into Mage1.5 CHO cells (Eureka, Calif.), special IgGglycoforms are produced. Similarly mouse VH and VL sequences weregrafted onto human IgG1 and IgG4 frameworks to make the ch3F8-IgG1 andch3F8-IgG4 recombinant antibodies.

Purification of hu3F8 and ch3F8 Hu3F8 and ch3F8 producer lines werecultured in Opticho serum free medium (InVitrogen, Calif.) and themature supernatant harvested. Protein A affinity column waspreequilibrated with 25 mM sodium citrate buffer with 0.15 M NaCl, pH8.2. Bound hu3F8 was eluted with 0.1 M citric acid/sodium citratebuffer, pH 3.9 and alkalinized (1:10 v/v ratio) in 25 mM sodium citrate,pH 8.2. It was passed through a Sartobind-Q membrane and concentrated to5-10 mg/ml in 25 mM sodium citrate, 0.15 M NaCl, pH 8.2. Stabilitystudies were performed on hu3F8-IgG1 in 25 mM sodium citrate 0.15 M NaClpH 8.2 versus PBS pH 7.4 in the presence or absence of 0.7 mg/ml oftween 80 (Sigma).

SDS-PAGE 2 ug each of the proteins is analyzed by SDS-PAGE undernonreducing or reducing conditions using 4-15% Tris-Glycine Ready GelSystem (Bio-Rad, Hercules, Calif.). Invitrogen SeeBlue Plus2 Pre-StainedStandard was used as the protein molecular weight marker. Afterelectrophoresis, the gel was stained using PIERCE's GelCode Blue StainReagent. The gel was scanned using Bio-Rad Fluor-S Multilmager(Bio-Rad), and the band intensity quantified with Quantity One software(Bio-Rad).

Quantitation of hu3F8 and ch3F8 by ELISA Microtiter plates were coatedwith GD2 at 20 ng per well. 150 ul per well of 0.5% BSA in PBS (diluent)was added to each plate for at least 30 min at ambient temperature toblock excess binding sites, the washed at least three times with PBS. Apurified batch of hu3F8-IgG1 (stock conentration 1 mg/ml) was used toconstruct a standard curve starting with 0.5 ug/ml followed by two folddilutions. 100 ul of standard and samples (also diluted 2-fold) wereadded to each well and incubated for 2.5 hours at 37° C. After washingplates 5 times with PBS, 100 ul of goat anti human-IgG (H+L) (JacksonResearch Laboratory) diluted at 1:3500 in diluent added to each well andincubated for 1 hour at 4° C. ELISA color reaction was developed withchromogen OPD (Sigma) with the substrate hydrogen peroxide for 30 min atRT in the dark. The reactions were stopped with 5N H₂S0₄and the OD readwith ELISA plate reader MRX (Dynex) at 490. Based on the standard curve,quantitation of hu3F8 supernatants was calculated in ug/ml or ug/mg ofprotein.

In vitro binding kinetics on Biacore T-100 biosensor (Biacore AB of GEHealthcare, Uppsala, Sweden) CM5 sensor chip (Research grade) andrelated reagents were purchased from Biacore USA (Piscataway, N.J.). Thegangliosides GM1 was from ALEXIS Biochemicals (AXXORA LLC, San Diego,Calif.), and GD2 from Advanced ImmunoChemical (Long Beach, Calif.). GM1was dissolved (0.5 mg/ml) in 90% ethanol, 10% methanol (v/v) and GD2 wasdissolved (0.5 mg/ml) in ethanol. Gangliosides were directly immobilizedonto the CM5 sensor chip via hydrophobic interaction. Reference surfacewas immobilized with GM1. GM1 was 1:1 diluted with 100% ethanol and thenwas 1/5 diluted in HBS-E buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 3mM EDTA). Diluted GM1(50 μg/ml) was injected (300 μl) at a flow rate of15 μl/min over 20 min. Extensive washing was followed with 10 mM NaOH(typically five washes of 20 μl at a flow rate of 5 μl/min) until astable baseline was obtained. Active surface was immobilized with GD2and GM1 in 1:1 ratio. GD2 and GM1 were 1:1 diluted with 100% ethanol andmixed in 1:1 ratio. The mixture of GD2 and GM1 was ⅕ diluted in HBS-Ebuffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 3 mM EDTA). Dilutedmixture of GD2 and GM1(50 μg/ml) was injected (300 μl) at a flow rate of15 μl/min over 20 min. Extensive washing was followed with 10 mM NaOH(typically five washes of 20 μl at a flow rate of 5 μl/min) until astable baseline was obtained.

Purified Anti-GD2 MoAbs were diluted in HBS-E buffer containing 250 mMNaCl at varying concentrations (50 ˜1600 nM) prior to analysis. 2.Samples (60 μl) were injected over the sensor surface at a flow rate of30 μl/min over 2 min. Following completion of the association phase,dissociation was monitored in HBS-E buffer containing 250 mM NaCl for300 sec at the same flow rate. At the end of each cycle, the surface wasregenerated using 50 μl 20 mM NaOH at a flow rate of 50 μl/min over 1min and 100 μl 4M MgCl2 at a flow rate of 50 μl/min over 2 min. Thebiosensor curves obtained following injection of the samples overimmobilized GD2 were subtracted with the control curves obtained withthe samples injected over immobilized GM1 prior to kinetics analysis.The data were analyzed by the bivalent analyte model and defaultparameter setting for the rate constants using the Biacore T-100evaluation software, and the apparent association on rate constant (kon,ka1), dissociation off rate constant (koff, kd1) and equilibriumdissociation constant (KD=kd1/ka1) were calculated.

Hu3F8 and ch3F8 were further characterized with rat anti-3F8anti-idiotypic antibodies (Cheung et al., 1993, Int J Can 54, 499-505).These rat IgG1 antibodies were also digested into Fab fragments andpurified using Fab preparation kit (Pierce Protein Research Products,Thermo Fisher Scientific). Their reactivities of ch3F8 and hu3F8 wereassayed by ELISA and by BIACORE.

ELISA for Cross Reactivity with Other Gangliosides GD2, GM2, GD1a, GD1b,GT1b, as well as GD3, GM3, GM1, GD1a were coated at 20 ng per well in90% ethanol. Following air drying, wells were blocked with 0.5% BSA inPBS at 150 ul per well for 1 hour at RT before washing for 3 times inPBS. Antibodies were added in triplicates at 1 ug/ml (100 ul per well)in 0.5% BSA. For background subtraction, wells with (1) no antigen and(2) no sample were used. Following incubation for 2 hour at 37° C. andwashing with PBS 5 times, HRP-goat anti mouse IgG (Jackson Laboratory,diluted to 1:1000) for mouse antibodies (e.g. 3F8) or HRP-goat antihuman IgG (Jackson Laboratory, 1:1000) for humanized antibodies wereused. Following additional incubation for 1 hour at 4° C. and furtherwashing, the OD was read using ELISA plate reader MRX (Dynex) at 490 andcross reactivity expressed as % maximal binding to GD2.

Biotinylation of Antibody Biotin (Long Arm)-N-hydroxysuccinimide ester(BNHS, Vector Laboratories, Inc., Burlingame, Calif.) was dissolved indimethylsulfoxide (DMSO) at a concentration of 50 mg/ml. Thebiotinylating reagent is added at 1/10 weight ratio of reagent to theantibody. With occasional stirring, the reaction mixture was incubatedat RT for 2 hours. The biotinylated antibody was dialyzed in PBS at RTfor 4 hours or at 4° C. overnight. The immunoreactivity of biotinylatedantibody was compared to the native antibody and ensured that EC50 bewithin 20% of each other.

Tissue cross-reactivity by immunostaining 5-7 micron thick frozensections were cut using cryostat, fixed with 250 ul of acetone at −20°C. for 30 min. After washing slides with PBS, slides were exposed tofreshly prepared 0.1% hydrogen peroxide for 15 min at RT. After washing,blocking Avidin solution (VECTOR Avidin-Biotin blocking kit) was addedand incubated for 20 min at RT. After washing, a drop of a blockingBiotin solution (VECTOR Avidin-Biotin blocking kit) was added andincubated for 20 min at RT. After further washing, >100 ul blockingserum (10% horse serum freshly diluted in PBS) was added and incubatedfor 1 h at RT. This step can be longer. Note: Must not reuse serum.After aspirating the blocking serum from each slide, 100 ul of 1 ug/mlbiotinylated MOPC21 (negative control) or biotinylated antibody dilutedin 1% horse serum was added and incubated for 1 hour at RT. Afterwashing, 100 ul of Avidin-Biotin Complex [ABC] (Vectastain ABC kit,VECTOR) at a 1:100 dilution in PBS was added and incubated for 30 min atRT. After washing, 200 ul of the dye (DAB Peroxidase Substrate kit,VECTOR) was added to each section and color allowed to develop for 2 min(until the desired intensity of staining was achieved based on colordevelopment in the standard). Sections were washed in running tap waterfor 5 min and counterstained with stock Myer's hematoxylin, and furtherwashing done in running tap water for 5 min. Slides were dehydratedsequentially in 75%, then 95%, and then 100% ethyl alcohol. Finaldehydrating step was in xylene or xylene substitute. One drop of freshCytoseal was added, and then section sealed with cover slip.

Direct cytotoxicity Antibodies were tested for their direct effect ontumor cell growth and survival in the absence of human serum or humaneffector cells. Tumor targets were dissociated with 2 mM EDTA orTrypsin-EDTA, washed and plated into 96-well flat bottomed plates in F10at 1.2×10³ to 3.5×10⁴ per well. After incubation for 24 hours in a CO2incubator at 37° C., 5% CO2, increasing concentrations of antibodies inF10 are added to each well. Control wells received F10 alone. Afterincubation for 72 hours at 37° C. in 5% CO2, WST-8 reagent was added toeach well and incubated in the dark in a CO2 incubator at 37° C. for 2-6hours. OD was read at 450 nm and 690 nm using ELISA plate reader MRX(Dynex). WST-8 assay was validated using direct cell counting usingTrypan Blue (Sigma) or Beckman Coulter Counter (Beckman Coulter, Brea,Calif.).

Antibody dependent cellular cytotoxicity (ADCC) by ⁵¹Chromium ReleaseTarget cells were detached with 2 mM EDTA in Ca2+ Mg2+ free PBS andwashed in 10% calf medium (Gibco) in RPMI 1640 (F10). 100 μCi of ⁵¹Crwas inucubated with 10⁶ target cells to in a final volume of 250 μl andincubated for 1 hr at 37oC with gentle resuspension of pellet at15-minute intervals. Cells were then washed and resuspended in 250 ulF10 and incubated for 30 min in 37° C. After washing, cells were countedand viability determined with trypan blue (Sigma) and quickly platedonto 96 well U-bottom plates. Peripheral blood from normal volunteerswere collected into heparinized tubes. The blood was mixed with 3%dextran/PBS and kept at RT for 20 minute to sediment the red cells.White cells were then ficolled and separated into peripheral bloodmononuclear cells (PBMC) and granulocytes (PMN) for PBMC-ADCC andPMN-ADCC, respectively. Cells were washed in F10, counted and viabilitydetermined. PBMC-ADCC was done in the presence of 10 U/ml of IL-2 andPMN-ADCC in 2 ng/ml of GMCSF. Final volume of ADCC was 250 ul/well.Antibodies were diluted in F10 from 1 ug/ml in 3-fold or 5-folddilutions. Plates were centrifuged at 500 rpm for snap spin at RT andthen incubated in a 37° C., 5% CO2 incubator for 4 hrs. Released ⁵¹Cr inthe ADCC supernatant was collected for gamma counting. Total release wasdetermined using 10% SDS and bakcgroun spontaneous release wasdetermined with F10 only without effectors. A E:T ratio of 50:1 wasgenerally used. Similarly, ADCC assays were carried out using NK92-MIcells stably transfected with the human CD16 or human CD32 Fc receptors.Unlike PBMC or PMN, no cytokines were needed in the assay. E:T ratio wasgenerally kept at 20:1.

${\% \mspace{14mu} {specific}\mspace{14mu} {lysis}} = {\frac{\left( {{experimental} - {background}}\; \right)}{\left( {{total} - {background}}\; \right)} \times 100\%}$

Iodination of antibodies Iodination using ¹²⁴I and ¹³¹I was carried outusing the iodogen method (Divgi et al., 2007, Lancet Oncol. 8, 304-10).A glass vial was coated with 50 μg of iodogen and 100 μg of IgG in 50 mMphosphate buffer (pH 7.4) was added together with 1 mCi of ¹²⁴I (or¹³¹I). Iodine 124 was produced on site by cyclotron at MSKCC. 10 mCi of¹²⁴I (0.02-0.05 mL) in 0.05 M NaOH was mixed with 1 mg of MoAb (˜0.5mL), 0.063 mL 1 M Tris base and 0.4 mL 0.2 M sodium phosphate pH 7.4 inthe reaction vial. The reaction was allowed to proceed on ice for 15minutes before the solution was removed and purified by size exclusionchromatography with a P6 size exclusion column and an eluant of 1% BSAin PBS.

Radioimmunoassay (RIA)

The method of Lindmo was used to estimate immunoreactivity (Lindmo etal., 1984, J Immunol Methods 72, 77-89). Radiolabeled MoAb was dilutedwith 1%BSA until 50 μl contained about 15,000-20,000 cpm (approximately1-1.5 uCi/3 ml). 50 μl was added to 500 μl 0.5% BSA containing 6.25,3.75, 2.5, 2.2, 1.9 million tumor cells and mixed for 1 hour at ambienttemperature. After centrifuge at 1500 rpm for 5 minutes the supernatantwas removed and washed with 1 ml ice cold 0.5% BSA. Following anothercentrifugation at 1500 rpm for 5 minutes, the cell pellets were countedin the gamma counter. Background counts in the absence of cells oriodinated antibodies were substracted and immunoreactivity estimated bythe Lindmo method (Lindmo et al., 1984, supra).

Biodistribution of MoAb in Xenografted Mice

Athymic nude mice xenografted with sc LAN1 neuroblastoma tumors wereused to study pharmacokinetics/biodistribution and anti-tumorproperties. Tumors were measured with a caliper. Experiments commencedwhen sc tumors reached ˜200 mg. ˜100 uCi of radioiodinated antibody permouse was injected intravenously and animals sacrificed usually at 48hours, and their organs removed and counted in a gamma counter (PackardInstruments, Perkin Elmer). These organs included skin, liver, spleen,kidney, adrenal, stomach, small intestine, large intestine, bladder,femur, muscle, tumor, heart, lung, spine, and brain. Based on the uCiaccumulated in the organ and the organ weight, % injected dose (ID)/gmof mouse was calculated. Tumor to normal tissue ratios of % ID/gm werealso calculated.

Sugar Analysis

Monosaccharide and oligosaccharide analyses of antibodies were carriedby the Complex Carbohydrate Research Center, Athens, Ga. Monosaccharideanalysis was assayed by HPAEC. N-glycan profiling was carried out byMALDI-MS.

EXAMPLE 1

Amino acid sequences of chimeric-IgG1 and four humanized IgG1. The CDRsof the heavy and light chains of m3F8 were grafted onto human IgG1frameworks based on their homology with human frameworks IGG HV3-33 andIGKV3-15, respectively. From two heavy chain and two light chaindesigns, four versions of hu3F8 were gene synthesized and expressed inDG44 cells. The amino acid sequences of chimeric human heavy and lightchains are shown SEQ ID NO:1 and 2, respectively, humanized heavy chainsequence huH1-gamma1 in SEQ ID NO:4, huH2-gamma1 in SEQ ID NO:6, andhumanized light chain sequences huL-1-kappa in SEQ ID NO:5 andhuL2-kappa in SEQ ID NO:7.

When expressed as whole IgG, the four hu3F8-IgG1 are namedhu3F8-H1L1-IgG1, hu3F8-H1L2-IgG1, hu3F8-H2L2-IgG1 and hu3F8-H2L1-IgG1.Additional constructs were made replacing the heavy chain sequences ofm3F8 and hu3F8-H1L1-IgG1 with the human IgG4 framework (chimeric heavychain gamma4, SEQ ID NO:4 and humanized 3F8 heavy chain gamma 4, SEQ IDNO:8) transfected into DG44 cells using the bluescript vectors. Inanother set of experiments, the hu3F8-H1L1-IgG1 sequence was transfectedinto a special MAGE1.5 CHO cell line selected for a specialglycosylation signature (See Materials and Methods). Both chimeric andhumanized 3F8 were purified using standard protein A affinitychromatography.

On SDS gel, chimeric and humanized antibodies migrated as IgG with theappropriate size heavy and light chains; and by HPLC they all eluted aswhole IgG with <10% aggregate formation (data not shown). By ELISA theyall bound to GD2 with similar avidity. By FACS analysis (LAN-1, data notshown) antibodies showed an optimal antibody concentration (˜0.1-1ug/million cells) beyond which mean fluorescence intensity (MFI) droppedbecause of cell death at higher antibody concentrations. With M14melanoma, cell death did not occur with excess antibody beyond 1 ug permillion cells (data not shown). IgG4 tended to have lower MFI because ofpreferential reactivity of the fluorescent second antibody with humanIgG1. Compared to the other hu3F8 constructs, hu3F8-H1L1-IgG1 was themost stable after freezing and thawing, retaining its binding to tumorcells by FACS and by ELISA (data not shown). By sugar analysishu3F8-IgG1n had mostly mannose and some N-acetyl-glucosamine, incontrast to hu3F8-IgG1 and hu3F8-IgG4 which contained near equal molarratios of fucose and N-acetyl-glucosamine (table 1).

EXAMPLE 2

Avidity Measurements by SPR (BIACORE)

With GD2 coated onto CM5 chips, kinetics of antibody binding (kon, koffand KD) were compared by Surface Plasmon Resonance using BIACORE T-100at low GD2 density at low GD2 density (table 2). Both k_(off) and K_(D)were improved proportionally for all antibodies at high GD2 density(data not shown). The Sensorgrams of representative antibodies atdifferent antibody concentrations were compared at both low GD2 density(data not shown) and at high GD2 density (data not shown) on the BIACORECM5 chips.

TABLE 1 Monosaccharide composition Antibody hu3F8- hu3F8- hu3F8- IgG1nIgG1 IgG4 Residue mole % mole % mole % Arabinose (Ara) 0 0 0 Ribose(Rib) 0 0 0 Rhamnose (Rha) 0 0 0 Fucose (Fuc) 0 15.1 13.2 Xylose (Xyl) 00 0 Glucuronic Acid (GlcUA) 0 0 0 Galacturonic acid 0 0 0 (GalUA)Mannose (Man) 95.2 44.1 49 Galactose (Gal) 0 0 0 Glucose (Glc) 0 0 0 NAcetyl Mannosamine 0 (ManNAc) N Acetyl Galactosamine 0 0 0 (GalNAc) NAcetyl Glucosamine 4.8 40.8 37.8 (GlcNAc) Heptose (Hep) 0 0 0 3Deoxy-2-manno-2 0 0 0 Octulsonic acid (KDO) Sum 100 100 100

The slow k_(off) of antibodies translated into a slower washoff whenantibodies were reacted with GD2-postive tumor cells. Here LAN-1 cells(data not shown) or M14 cells (data not shown) were reacted withmonoclonal antibodies and washed multiple times in wash buffer. Witheach wash, the remaining antibodies on the cell surface were detectedusing a secondary FITC-labeled goat anti-mouse antibody. MFI wasexpressed as a percent of the baseline (i.e. after first wash). On LAN-1cells, 3F8 and anti-B7-H3 8H9 antibodies had slow wash off (˜80%retention after 10 washes) compared to 14.G2a (˜30% retention, data notshown). Similarly for M14 tumor cells, by the 5^(th) wash, there wassubstantial difference in the retention of antibody on tumor cells.While 3F8, 3F8-(Fab′)2, and hu3F8-IgG1 (all with slow koff) had >80%retention, other Anti-GD2 antibodies 14.G2a (mIgG2a), ME361 (mIgG2a),and S220-51 (mIgG3) (all with fast koff by BIACORE) leaked off to <30%.Hu3F8-IgG1, hu3F8-IgG4, ch3F8-IgG1 and ch3F8-IgG4 also reactedspecifically to rat anti-3F8 antiidiotypic antibodies (Cheung et al.,1993, Inter J Cancer 54, 499-505) and their Fab fragments with highavidity in ELISA and by SPR (See Methods).

EXAMPLE 3

Crossreactivity with other Antigens

In cross reactivity studies, hu3F8-H1L1-IgG1 had similar profile asch3F8-IgG1 and m3F8 (table 3). There was low level of cross reactivitywith GD1b expressed as percent OD by ELISA relative to the OD on solidphase GD2. There was no cross reactivity of m3F8, hu3F8 or 14.G2a withhuman N-CAM (Patel et al., 1989, Br. J. Cancer 60, 861-866) either byWestern blots or by SPR (data not shown).

TABLE 2 Binding kinetics of chimeric and humanized 3F8 by SPR(BIACORE-T100) Antibody n kon koff KD (nM) ch3F8-IgG1 2 1.15E+051.45E−03 13 ± 3  hu3F8-H1L1-IgG1 12 9.19E+04 1.03E−03 11 ± 1 hu3F8-H1L2-IgG1 3 1.74E+05 1.07E−03 7 ± 2 hu3F8-H2L1-IgG1 3 1.92E+055.04E−03 31 ± 14 hu3F8-H2L2-IgG1 3 1.52E+05 3.51E−03 26 ± 11hu3F8-H1L1-IgG1n 3 9.76E+04 6.88E−04 11 ± 2  ch3F8-IgG4 2 9.40E+041.28E−03 14 ± 2  hu3F8-H1L1-IgG4 3 1.18E+05 1.76E−03 15 ± 1  m3F8 101.75E+05 1.04E−03 5 ± 1 m3F8 (Fab′)2 3 1.44E+05 1.23E−03 9 ± 3 14.G2a 61.30E+05 1.11E−02 100 ± 26 

EXAMPLE 4

Direct Cytotoxicity

When these antibodies were added to neuroblastoma cells in vitro, theyinduced direct cell death and slowed down in vitro cell growth. Whenassayed by WST-8 in a 3-day culture system, m3F8 and hu3F8 had similarpotency when their EC50s were compared (see table 4), in contrast to14.G2a which was ˜10-fold weaker.

TABLE 3 Cross reactivity with other gangliosides by ELISA # GM2/ GD1a/GD1b/ GT1b/ GQ1b/ Antibody exp GD2 GD2 GD2 GD2 GD3/GD2 GD2 14.G2a 7 0 00 0 0 0 m3F8 19 0 0 4.1% 0 0 0 ch3F8-IgG1 5 0 0.2% 8.8% 0 0.2% 0ch3F8-IgG4 6 0 0 6.1% 0 0 0 hu3F8-H1L1-IgG1 44 0 0 4.5% 0 0 0hu3F8-H1L2-IgG1 4 0 0 2.9% 0 0 Nd hu3F8-H2L1-IgG1 8 0 0 1.9% 0 0 0hu3F8-H2L2-IgG1 9 0 0 1.6% 0 0 0 hu3F8-H1L1-IgG4 11 0 0 3.1% 0 0 0hu3F8-H1L1-IgG1n 6 0 0 6.9% 1.0% 0 0

EXAMPLE 5

Antibody Dependent Cell Mediated Cytotoxicity

The four hu3F8 IgG1 antibodies (H1L1, H1L2, H2L1, H2L2) were compared inADCC assays using volunteer PBMC (data not shown) and volunteer PMN(data not shown) as effectors. Other than H1L2, all three hu3F8 hadcomparable PBMC-ADCC. But most importantly, they were all superior tom3F8 by a factor of >10-100.

TABLE 4 Direct cytotoxicity of neuroblastoma LAN-1 in the presence ofantibodies Direct Cytotoxicity Antibody EC50 (ug/ml) m3F8 1.9hu3F8-IgG1-H1L2 10.5 hu3F8-IgG1-H2L1 16.5 hu3F8-IgG1-H2L2 17.5hu3F8-IgG1-H1L1 5.1 hu3F8-IgG1n 2.6 hu3F8-IgG4 3.1 ch3F8-IgG1 4.5ch3F8-IgG4 6.4 14.G2a 47.1

Hu3F8-H1L1-IgG1 (abbreviated as hu3F8-IgG1) was the most stable in vitroand was chosen for further characterization. Ch3F8-IgG1, hu3F8-IgG1,hu3F8-IgG1n, 14.G2a, and m3F8 were compared in ADCC assays against LAN-1using PBMC (data not shown) or PMN (data not shown) as effectors. TheADCC potencies of these antibodies were computed as the ratio (EC50 for3F8)/(EC50 for MoAb) (table 5). Relative to m3F8, hu3F8-IgG1 was 217fold stronger in PBMC-ADCC, and 19 stronger in PMN-ADCC. Forhu3F8-IgG1n, it was 3901 fold and 5 fold, respectively. In addition, themaximal cytotoxicity achieved with both chimeric and humanized 3F8 weresubstantially and consistently higher than that of m3F8 or 14.G2a,irrespective if it was PBMC-ADCC or PMN-ADCC.

In order to examine the capability of MoAb in ADCC for individual FcR inisolation (in the absence of inhibitory FcR), we tested ADCC using NK92cells. NK92 cells do not carry human FcR on their cell surface. Whentransfected with human CD16 and CD32, they could mediate efficient ADCC.When these effector cells were tested against neuroblastoma LAN-1targets, hu3F8-IgG1n was substantially (˜10 fold) more efficient thanhu3F8-IgG1, which was in turn more efficient (12 fold) than m3F8 inCD16-ADCC (data not shown). In contrast, with CD32 as FcR, the potencywas nearly 10-fold higher for hu3F8-IgG1 compared to that of hu3F8-IgG1n(data not shown). Similar trends were observed when melanoma M14 wasused as targets. Hu3F8-IgG1n was ˜10-fold more efficient than hu3F8-IgG1or ch3F8-IgG1, which were in turn ˜20-fold more efficient than m3F8 or14G.2a (data not shown) in CD16-ADCC. In contrast, hu3F8-IgG1,hu3F8-IgG1n and ch3F8-IgG1 were similar when CD32 was the FcR,all >10-fold more efficient than m3F8 in CD32-ADCC (table 6). IgG4subclass antibodies had <3% of CMC activity, minimal CD16-ADCC activity,but better C32-ADCC than m3F8.

EXAMPLE 6

Complement Mediated Cytotoxicity

In human complement mediated lysis (CMC), the different hu3F8 IgG1 forms(H1L1, H1L2, H2L1, and H2L2) were comparable in efficiency (table 6).Ch3F8 and hu3F8 were 40-60%, while 14G.2a and ch14.18 were 4-12% asefficient in CMC as m3F8 (table 6).

TABLE 5 Relative antibody potency in ADCC and CMC against neuroblastomaLAN-1 LAN1- LAN1- LAN1- LAN1- LAN1- PBMC PMN CMC CD16 CD32 AntibodyPotency Potency Potency Potency Potency 14.G2a 0.03 1.0 0.12 4 2 ch14.1811 3 0.04 2 8 ch3F8-IgG1 390 18 0.64 24 13 ch3F8-IgG4 0 1 0.01 0 3hu3F8-IgG1 217 19 0.40 12 15 hu3F8-IgG1n 3901 5 0.41 106 2 hu3F8-IgG4 04 0.03 0 1 m3F8 1 1 1 1 1 hu3F8-H1L2- 7 11 0.22 12 17 IgG1 hu3F8-H2L1-355 14 0.64 1 7 IgG1 hu3F8-H2L2- 390 15 0.47 10 3 IgG1

EXAMPLE 7

Targeting Human Neuroblastoma Xenografts

Hu3F8-IgG1, hu3F8-IgG1n, and hu3F8-IgG4 were radiolabeled with ¹³¹I withcomparable immunoreactivity of around 40-45% (Table 7). Theirbiodistributions at 48 hours were compared with that of ¹³¹I-m3F8 inmice bearing sc LAN-1 neuroblastoma xenografts. Tumor uptake whenmeasured by % ID/gm was comparable between hu3F8-IgG1 (29.6%) and m3F8(28.6%), both nearly double those of hu3F8-IgG1n and hu3F8-IgG4 (datanot shown). Tumor to normal tissue ratios were comparable among these 4antibodies for NB LAN-1 xenografts. In comparison, with melanoma M14both the % ID/gm and the Tumor to normal tissue ratios of all threehu3F8 antibodies were similar to that of m3F8 (data not shown).

TABLE 6 Relative antibody potency in ADCC and CMC against Melanoma M14M14-CD16 M14-CD32 Antibody Potency Potency 14.G2a 1 6 ch14.18 9 21ch3F8-IgG1 39 17 ch3F8-IgG4 0 0.5 hu3F8-IgG1 20 31 hu3F8-IgG1n 252 10hu3F8-IgG4 0.1 4 m3F8 1 1 hu3F8-H1L2-IgG1 40 44 hu3F8-H2L1-IgG1 6 9hu3F8-H2L2-IgG1 15 11

EXAMPLE 8

Treatment of Neuroblastoma Xenografts

Mice xenografted with established human neuroblastoma LAN-1 (0.5-1 cmdiameter) were treated with iv m3F8 or hu3F8-H1L1-IgG1 twice weekly for4 weeks. Tumor response and mice survival were monitored. Delay in tumorgrowth was dependent on hu3F8-H1L1-IgG1 antibody dose (200 ug>100 ug>20ug, data not shown). Survival of mice receiving 100-200 ug weresignificantly longer (p=0.003) than mice receiving PBS control or m3F8(data not shown).

TABLE 7 RIA of 131I-labeled antibodies in biodistribution studies LAN1m3F8 hu3F8-IgG1 hu3F8-IgG1n hu3F8-IgG4 RIA 45% 45% 42% 40% TCA 94% 96%96% 96% # mice 18 19 17 19

Discussion

Anti-GD2 antibody is a proven therapy for GD2-postive neuroblastoma (Yuet al., N Engl J Med 363:1324-1334, 2010). The murine antibody 14.18 andits derivatives (14.G2a and ch14.18) have provided benchmarks for futureimprovements of anti-GD2 therapy. We chose murine IgG3 antibody m3F8 forclinical development because of its 10-fold slower koff during GD2binding compared to 14.G2a or ch14.18. Among patients withchemoresistant metastatic neuroblastoma in the bone marrow, m3F8 plusGMCSF induced 80% complete remissions (Kushner et al., 2001, J ClinOncol 19, 4189-94). However, human anti-mouse antibodies (HAMA) candiminish the effect of the murine antibody by neutralizing its abilityto bind to its antigen, by blocking the direct effect of the antibodyand by accelerating the clearance of the antibody from circulation.Genetic engineering to change murine to human IgG frameworks shouldreduce the HAMA response. Ch14.18 and hu14.18 (both derived from the VHand VL of 14.G2a) have minimal immunogenicity in patients. We thereforetested the chimeric and humanized forms of 3F8 as potential nextgeneration Anti-GD2 antibodies

One criterium for successful chimerization and humanization is thepreservation of affinity during genetic engineering. This isparticularly uncertain with CDR grafting in the humanization process.The preservation of a slow koff in ch3F8 and hu3F8 was reassuring. Butmore importantly, the preservation and enhancement of in vitro effectorfunction, as well as in vivo tumor targeting plus in vivo therapeuticproperties could be critical. Both ch3F8-IgG1 and hu3F8-IgG1 showed >200fold more efficient PBMC-ADCC than m3F8, while PMN-ADCC were >20 fold.In addition, the special glycoform hu3F8-IgG1n had >3500 fold moreefficient PBMC-ADCC, and 5 fold more efficient PMN-ADCC than those ofm3F8. In sharp contrast, for CMC, ch3F8 and hu3F8 had lower complementactivating ability than m3F8.

This large improvement in ADCC is most desirable given recent evidencefor its role in the anti-tumor effects of monoclonal antibodies inpatients. Among lymphoma patients treated with rituximab, both highaffinity FcR2A and FcR3A were shown to have better response and survivaladvantage (Weng and Levy, 2003, J Clin Oncol 21, 3940-7). While the highaffinity receptor FcR3A translated into <10 improvement in ADCC in vitro(Niwa et al., 2004, Clin Cancer Res 10, 6248-55), overall response andtime to progression improved by 200% (Weng and Levy, 2003, supra). Whenmetastatic breast cancer was treated with Herceptin, patients with highaffinity FcR3A had better overall response (83% versus 35%, p=0.03), andlonger progression-free survival (p=0.005)(Musolino et al., 2008, J ClinOncol 26, 1789-96). For metastastic colorectal cancer treated withCetuximab, patients with low affinity FcR2A and FcR3A had comparablehazard ratios as patients with mutated KRAS (Bibeau et al., 2009, J clinOncol 27, 1122-9). With murine 3F8, patients with the high affinityFcR3A receptor for m3F8 on myeloid cells were shown to have bettersurvival (Cheung et al., 2006, J Clin Oncol 24, 2885-90).

While binding affinity and effector functions are critical fortherapeutic applications, cross-reactivity can pose unexpected toxicityissues. We showed that these chimeric and humanized forms for 3F8 hadcomparable cross-reactivity patterns as the murine 3F8 both by ELISAassays with purified gangliosides, as well as by immunohistochemistry ona panel of normal humans tissues (data not shown). Similar to m3F8, bothchimeric and humanized forms of 3F8 showed low levels of reactivity toGD1b when compared to GD2. GD1b has been shown to be highly prevalentganglioside among neuroblastoma tumors, especially when differentiatedby retinoids (Hettmer et al., 2004, Br J Cancer 91, 389-97; Gong et al.,2002, Brain 125, 2491-506). This maybe relevant since cis-retinoic acidis routinely given to patients undergoing immunotherapy for high-riskneuroblastoma. However, anti-GD1b antibodies have also been associatedwith sensory ataxic neuropathies (Gong et al., 2002, supra).Nevertheless, the safety profile of m3F8 (with similar cross reactivityto GD1b) with no permanent or late sensory neuropathies in more than 500patients was reassuring.

Complement-mediated cytotoxicity (CMC) is unusually effective againsthuman neuroblastoma (Saarinen et al., 1985, Proc Amer Assoc Cancer Res26, 291) because of their low expression of CD55 (Cheung et al., 1987,Proc Natl Am Assoc Cancer Res 28, 387) and CD59 (Chen et al., 2000,supra). All Anti-GD2 antibodies mediate efficient complement mediatedcytotoxicity, and m3F8 seems particularly efficient. Yet, studies ofrituximab have suggested a negative role of complement activation indown regulating ADCC (Wang et al., 2009, Blood 114, 5322-30). Inclinical studies, higher activity of complement component C1qA wasassociated with less favorable response to rituximab therapy (Racila etal., 2008, Clin Cancer Res 14, 6697-703). For anti-GD2 antibodies,complement activation was thought to be responsible for the pain sideeffects (Navid et al.,Curr Cancer Drug Targets 10:200-209, 2010) hencethe Fc-CH2 domain mutated version (hu14.18K322A) currently in clinicaltrial. Given these considerations, an overdrive of CMC is probably notdesirable. It is reassuring that both ch3F8 and hu3F8 had slightly lessefficient CMC.

EXAMPLE 9

Using Antibody to Block acute Pain Side Effects

It was hypothesized that antibody with slow koff but depleted of ADCC orCMC activity could be used to block GD2 on nerve fibers surrounding theperivascular space. Heat treatment of 3F8 (HM3F8) depletes its ADCC andCMC activities without affecting its GD2 binding (Kushner et al. 2011, JClin Oncol 29, 1168-1174). When rats were pre-injected with heat-treated3F8, their pain response to a subsequent injection of 5-10 molar excessof native 3F8 was substantially reduced. More importantly, anti-tumoreffect was not compromised. In a phase I clinical trial (IRB-05015,NCT00450307), 30 patients with resistant neuroblastoma (NB) received oneto two cycles of 3F8 plus granulocyte-macrophage colony-stimulatingfactor in the outpatient setting. 3F8 dosing began at 20 mg/m²/d andincreased by 20 mg/m²/d in the absence of dose-limiting toxicity (DLT).Premedication included analgesics, antihistamines, and 5-minuteinfusions of 2 mg/m² HM3F8. Opioid use was compared with a contemporarycontrol group treated with 3F8 at 20 mg/m²/d but no HM3F8. Doseescalation stopped at 160 mg/m²/d because of drug supply limitations;even through this dosage level, analgesic requirements were similar tohistorical controls, and there were no DLTs. Analgesic requirements at3F8 dosage levels through 80 mg/m²/d were significantly less comparedwith controls. Anti-NB activity occurred at all dosages. This multifolddose escalation of 3F8 was feasible and suggested that HM3F8 couldmodify toxicity without blunting anti-NB activity.

Pain Blocking Potential of hu3F8-IgG4

Based on the clinical results of the HM3F8, we hypothesize thatperivascular pain fibers can be preferentially blocked by a small doseof an antibody devoid of CMC or ADCC function. Although heating of m3F8destroys its CMC and ADCC functions (while retaining binding), heatmodified hu3F8 retains near full CMC and ADCC functions. The inabilityof hu3F8-IgG4 to activate CMC and ADCC in different NB cell lines suchas Lan1, NMB7, SKNLP, BE(1)N and SHEP1(Table 8 and data not shown) makesthis antibody subclass a viable alternative for HM3F8. Unlike heatingwith unknown effects on antibody structure and immunogenicity,hu3F8-IgG4 is an engineered protein. Besides the absence of CMC and ADCCfunction, IgG4 subclass has unique biochemical properties. Typicallyinduced by chronic antigen stimulation, they are known to interfere withimmune complex formation by other antibody isotypes, thereby dampeningthe inflammatory reactions (Losen et al., 2008, Ann N Y Acad Sci 1132,174-9). Most importantly, it has a natural ability to reduce itself tomonovalency, thereby losing its ability to compete with hu3F8-IgG1 aftera period of hours in blood (van der Neut Kolfschoten et al., 2007,Science 317, 1554-7). Such monovalency derives from its known propertyto exchange Fab arms by swapping a heavy chain and attached light chain(half-molecule) with a heavy-light chain pair from another IgG4molecule, which results in monovalency towards the specific antigen inquestion (e.g. GD2).

TABLE 8 Antibody potency using NK92-CD16 ADCC as effectors on differentNB lines LAN1 NMB7 SKNLP BE(1)N SHEP1 14G2a 0.4 1 1 1 1 ch14.18 4 94 8 23 hu3F8-IgG1 26 1428 23 68 3 hu3F8-IgG4 0 0 0 0 0 m3F8 1 1 1 1 1 HM3F80.2 — — — —

Rat allodynia model The rat model was used to test the ability ofhu3F8-IgG4 to block pain side effects (MSK protocol #09-05-010). Bothm3F8 (an efficient activator of rat complement, Bergman et al., 2000,Cancer Immunol Immunother 49, 259-66) and hu3F8 were used to induceallodynia. Hu3F8-IgG4 (0.1, 0.25 or 0.5 mg/kg) was administered as ivpush 30 minutes prior to iv m3F8 (5 mg/kg), or hu3F8-IgG1 (5 mg/kg), orch14.18 (5 mg/kg). Other groups receive heat modified m3F8 (HM3F8) orcontrol antibodies instead of hu3F8-IgG4. Von Frey filaments method wasused to evaluate pain at baseline prior to and after MoAb injection(Chassaing et al., 2006, Br J Clin Pharmacol 61, 389-97; Benani et al.,2003, Eur J Neurosci 18, 2904-14).

Effect of hu3F8-IgG4 on biodistribution and efficacy of ¹³¹I-hu3F8-IgG1Neuroblastoma (LAN-1) tumors were planted subcutaneously in groups offemale NOD/SCID/IL2-gc^(nu11) immunodeficient mice (n=10 per group) withlow serum IgG. Mice were injected with human IgG at 1 gm/kg 24 h priorto experiment. Hu3F8-IgG4 (0.1, 0.25 or 0.5 mg/kg) or control huIgG4(0.1, 0.25 or 0.5 mg/kg), or HM3F8 (0.5 mg/kg), or saline wereadministered as iv push, 30 minutes prior to m3F8, or hu3F8-IgG1 orch14.18 (5 mg/kg) trace labeled with ¹³¹I-labeled respective MoAb (50uCi/mouse). Blood was collected from the tail at 1, 3, 6, 12, 24, 36, 48and 96 h. In biodistribution studies mice were sacrificed at 2 timepoints (24 h and 48 h after ¹³¹I-MoAb injection) for tissue counts. Themajor organs were removed and counted in a gamma counter with a knownvolume of the injectate and the data expressed as a percentage ID/gmtissue. Pharmacokinetics analysis was be carried out bynon-compartmental analysis of the serum concentration-time data usingthe WinNonlin software program (Pharsight Corp., Mountain View, Calif.).In separate sets of experiments, xenografted mice were treated similarlybut without ¹³¹I-trace label, and their sc tumor response measuredovertime for 2 months.

The ability of hu3F8-IgG4 to block allodynia in the rat can be comparedto that of heat-treated m3F8. Based on the dose response curves of theblocking antibody used (while keeping the challenge by native m3F8, orhu3F8-IgG1 or ch14.18, all at 5 mg/kg) a relative potency in blockingallodynia in the rat can be derived. We anticipate hu3F8-IgG4 to beequivalent or more efficient when compared to heat-treated 3F8 inblocking allodynia. These studies will provide preclinical rationale fortransitioning hu3F8-IgG4 to a clinical trial.

EXAMPLE 10

Optimization of hu3F8 Framework Sequence for Enhanced Stability

In the process of humanizing murine antibodies, framework residues atstructurally important positions are often back mutated to the murinesequence in order to preserve both antibody stability and antigenbinding (Honegger, A, 2008, Engineering Antibodies for Stability andEfficient Folding, In: Therapeutic Antibodies. Handbook of ExperimentalPharmacology, 181).

Materials and Methods

Structural Model of m3F8 Variable Domain

A homology model of m3F8 variable domain was created using MODELER(Eswar et al., 2006, Curr Prot Bioinfor, Supplement 15, 5.6.1-5.6.30) inDiscovery Studio (Accerlys, San Diego, Calif.). The crystal structure ofmalaria antigen AMA1 antibody Fab (Protein Data Bank code 2Q8A) waschosen as a template for the variable light chain (84% identity). Thecrystal structure of antibody 13G5 Fab (Protein Data Bank code 2GJZ) waschosen as the template for the variable heavy chain.

Crystal Structure of m3F8 Fab Fragment

Fab fragments of m3F8 were generated by papain digestion using astandard Fab preparation kit (Pierce Biotechnology, Rockford, Ill.). Thepurified m3F8 was concentrated to 12 mg/ml in 20mM HEPES pH 6.5 and wascrystallized in a hanging drop by vapor diffusion at 16° C. against areservoir containing Hampton Index reagent D9 containing 0.1M Tris pH8.5, 25% w/v PEG 3350 (Hampton Research, Aliso Viejo, Calif.). Thedroplet was formed by mixing 1 μl of protein solution and 1 μl ofreservoir solution. The crystals were protected by cryoprotectantcontaining 25% glycerol, 0.1M Tris pH 8.5, 25% w/v PEG 3350. Data wascollected at the Argonne Advanced Photon Source beamline 24IDC. Thecrystals belong to the space group C2 and diffract to 1.7 Å resolution.

The Fab structure was solved by molecular replacement using Phaser (CCP4suite) (McCoy et al., 2007, J Appl Crystallogr 40, 658-674) and PDBentry 2AJU as the search model (Qin et al., 2008, J Biol Chem 283,29473-84). The best molecular replacement model was refined usingRefmac5 (Murshudov et al., 1997, Acta Crystallogr. D: Biol. Crystallogr53, 240-255), manual fitting was performed with O (The CCP4 suite:programs for protein crystallography. Acta Crystallogr., D: Biol.Crystaollogr. 50 (1994) 760-763), adding solvent with Arp-Warp (Lamzinand Wilson, 1993, Acta Crystallogr, D: Biol. Crystallogr. 49, 129-147).The final model contained two polypeptide chains of m3F8 Fab and 585solvent molecules.

Computational Analysis of m3F8 Crystal Structure

Based on the human templates IgHV3-33-HC and IGKV3-15-LC, eachhumanizing mutation that would be introduced into the framework on m3F8was analyzed using computational chemistry methods. The crystalstructure of m3F8 was simulated using CHARMm (Chemistry at HarvardMolecular mechanics) force fields (Brooks et al, 2009, J. Comp Chem 30,1545-1615), and the effect of each point mutation was calculated fromthe difference between the folding free energy of the mutated structureand the wild type protein. Generalized Born approximation was used toaccount for the effect of the solvent and all electrostatic terms werecalculated as a sum of coulombic interactions and polar contributions tothe solvation energy. A weighted sum of the van der Waals,electrostatic, entropy and non-polar terms was calculated for each pointmutation. All calculations were performed using Discovery Studio 3.0(Accelrys, San Diego, Calif.).

Results

For the hu3F8-H1L1 heavy chain sequence, the human IgG heavy chainsequence IgHV3-33-HC was used as a template and back mutations were madeat positions 5, 9, 16, 19, 20, 24, 28, 29, 30, 40, 48, 49, 67, 71, 76,78, 81, 86, and 92. For the hu3F8-H1L1 light chain, the human IgG lightchain sequence IGKV3-15-LC was used as a template and back mutationswere made at positions 1, 7, 9, 15, 19, 21, 22, 43, 45, 58, 60, 67, 70,73, 78, 80, and 87. The back mutations were deduced based on a homologymodels of m3F8, and involved determining that these whether themutations would influence the folding of the CDR loops or affectbackbone stability (mutations involving Gly and Pro).

To verify the accuracy of the homology model and further enhance thehumanization strategy, the crystal structure of m3F8 was solved at 1.7 Å(data not shown). The homology model of m3F8 variable region wassuperimposed with the solved crystal structure (data not shown). Thehomology model for the majority of the framework region outside of theCDR closely matched the experimentally derived crystal structure.Notable differences were, however, observed in the CDR region,particularly in loops H3, H2, and L3. The homology model also failed toaccurately predict any of the interactions at the VH-VL interface thatare crucial to the stability of the Fv structure. There are 6 pairs ofamino acids that hydrogen bond at the VH-VL interface and two pairs ofamino acids that participates in pi-interactions (Table 10). After thecrystal structure of m3F8 was solved, computational analysis was appliedto determine the energetic effect of each humanizing mutations based onIgHV3-33-HC and IGKV3-15-LC templates. The difference in the foldingfree energy was calculated for each mutation. Based on this analysis,five of the humanizing mutations were found to be destabilizing to theoverall fold of m3F8 (see Table 9). These include heavy chain positions11 and 21, and light chain positions 1, 10, 12, 40, and 97. It was alsodetermined that humanizing mutations involving Pro and Gly in the lightchain at positions 40 and 97, respectively, would significantly affectthe backbone flexibility. These five destabilizing mutation in additionto the two Gly/Pro mutations were considered candidates for backmutation and are included in the sequence of hu3F8-H3L3 (SEQ ID NO:9 and10). The computational analysis of the m3F8 crystal structure alsoidentified five humanizing mutations that would have neutral affects toantibody stability (data not shown). These mutations are positions 24and 56 of the light chain and at positions 20, 58 and 92 of the heavychain. These mutations were not included in sequence of hu3F8-H1L1 (SEQID NO:4 and 5), which was based on the homology model, but are includedin the design on hu3F8-H3L3.

An analysis of the VH-VL interface of the crystal structure of m3F8 wasdone to preserve each of the hydrogen bonds and pi-interactions in hu3F8(Table 10). Of the 15 amino acids that are involved in interfaceinteractions, 14 were preserved in the design of hu3F8-H1L1 andhu3F8-H3L3. The one missing interaction involves Ser43 in the lightchain. This mutation was added to the design of hu3F8-L1 and hu3F8-L3 togenerate Hu3F8-L1S and Hu3F8-L3S.

These stability-enhanced frameworks would not have possible with thecurrent state of the art, which involves the use of homology modeling todesign stable humanized frameworks. The use of an experimentally derivedcrystal structure of m3F8 in combination with computational analysisusing force field methods were needed to design novel antibodies withenhanced stability profiles.

TABLE 9 Computational analysis of humanizing mutations on crystalstructure of m3F8 Fv Mutation ΔG (kcal/mol) Observation Mutations thatwill destabilize the mouse structure or alter backbone flexibility.Light chain S1E 0.82 Destabilizing Light chain F10T 1.36 DestabilizingLight chain L12S 1.98 Destabilizing Heavy chain L11V 1.90 DestabilizingHeavy chain T21S 1.71 Destabilizing Light chain A40P −1.03 Changesbackbone flexibility Light chain G97Q −1.42 Changes backbone flexibilityMutations that will have negligible affects on stability. Light chainK24R −0.30 Neutral effect Light chain S56T 0.24 Neutral effect Lightchain V58I −0.73 Neutral effect Heavy chain I20L −0.44 Neutral effectHeavy chain M92V −0.56 Neutral effect

TABLE 10 Important interactions between amino acids at the VH-VLinterface of m3F8 Fv crystal structure Light chain Heavy chainInteraction residue residue H-bond Gln38 Gln39 H-bond Ser43 Gly110H-bond Tyr55 Asp107 H-bond Gln89 Tyr104 H-bond Tyr92 Arg98 H-bond Tyr36Leu106 Pi-Pi Tyr92 Trp47 Pi-sigma Tyr92 Tyr104

EXAMPLE 11

Higher Affinity 3F8 Antibody Based on Computational Analysis of 3F8:GD2Interaction

The crystal structure of m3F8 was used as a template for computationalstudies to determine the molecular details of antigen recognition. TheGD2 antigen is composed of a penta-saccharide head group linked to aceramide lipid tail. In the absence of an experimentally derived 3F8:GD2co-complex, computational docking was attempted with the GD2penta-saccharide head group only, where the ceramide moiety was replacedby a methyl-group. The GD2 penta-saccharide molecule presented a majorchallenge for docking studies given the state of the field incomputational docking. Firstly, the docking of large flexible ligandsare difficult to accurately predict. The GD2 head group has over 30rotatable bonds. There is no established docking protocol that has beenreported to be accurate for a ligand with such a high degree offlexibility. Secondly, the GD2 head group is a carbohydrate molecule,and there are few reports on the accuracy of docking studies when forcefield methods are applied to carbohydrate molecules. Thirdly, the GD2head group is a special class of carbohydrates since it contains twocharged groups found in its sialic acid moieties. No established dockingmethods have been shown to accurately predict the binding conformationof such large, flexible, and charged carbohydrates.

One docking algorithms that has been reported to be reliable forpredicting the binding conformation of oligosaccharides (although notcontaining charged sialic residues) is GLIDE (Agostino et al., 2009, JChem Inf Model 49, 2749-60). The report showed that GLIDE outperformedAutoDock, GOLD, and FlexX in predicting the binding conformation ofoligosaccharides to antibodies as compared to experimentally derivedco-crystal structures. It should be noted that none of the antigenstested were larger a tetra-saccharide. Another promising dockingalgorithm in the literature was CDOCKER (Wu et al., 2003, J. Comp Chem24, 1549), a CHARMm based docking algorithm that was shown to outperformDOCK, FlexX, and GOLD in accurately predicting the docking conformationof ligands with 8 or more rotatable bonds (Erickson et al., 2004, J MedChem 47, 45-55). It should be noted that oligosaccharides were notanalyzed in the study, and the ligands tested were much smaller and lessflexible that the GD2 head group (>30 rotatable bonds).

A head-to-head comparison between GLIDE and CDOCKER was performed indocking ligands similar to GD2 (sialic acid containing oligosaccharides)using three available test cases from the protein data bank (PDB code2HRL: Siglec-7 in complex with GT1b; PDB code 3BWR: Simian virus VP1 incomplex with GM1; and PDB code 3HMY: Tetanus toxin HCR/T in complex withGT2). In all two of three test cases, CDOCKER was able to accuratelypredict the correct binding mode of the respective oligosaccharide(within 2 Ångstom root-mean-square deviation).

GLIDE inaccurately docked two to the three test cases (>2 Ångstomroot-mean-square deviation) and failed to find a docked pose in thethird test case. (see Table 11). By this analysis, CDOCKER outperformedGLIDE in docking charged oligosaccharides. GLIDE failed in each one ofthe cases (see Table 11). CDOCKER was then used to dock the GD2penta-saccharide head group to the antigen-binding pocket of the crystalstructure of m3F8. The top docked structure was then energy minimizedusing CHARMm force fields. The analysis indicated 14 amino acids thatdirectly interact with the GD2 head group (Table 12). Each of thesepositions was then mutated in silico to all of the possible amino acidsand the CHARMm based interaction energies were calculated. The highestinteracting mutants are listed in Table 13. Based on this approach, onemutation (Heavy Chain Gly54Ile) was predicted to increase theinteraction energy significantly. Computational modeling showed thatsubstitution of the Gly at position 54 to Ile in the CDR3 of the heavychain would change the shape of the binding pocket and increase thecontact with the GD2 ligand. This analysis was used to add the heavychain mutation Gly54Ile to the CDR regions of the sequences ofhuH1-gamma1 and huH3-gamma1 to create huH1I-gamma1 and huH3I-gammarespectively.

Docking Methods

GLIDE docking was performed using the Schrodinger Suite 2009(Schrodinger, New York, N.Y.). OPLS force fields were used toparameterize the proteins and ligands. Top ligand poses were clusteredwithin a root-mean-square deviation of 2.0 Å and scored by GlideScore.

CDOCKER docking and interaction energy measurements were performed usingDiscovery Studio 3.0 (Accelrys, San Diego, Calif.). CHARMm force fieldswere used to parameterize the proteins and ligands. Top ligand poseswere clustered within a root-mean-square deviation of 2.0 Å and scoredby CDOCKER Interaction Energy.

TABLE 11 Comparison of docking algorithms GLIDE versus CDOCKER inpredicting known protein:ganglioside complexes RMSD of top pose MethodProtein Ligand (Å) CDOCKER Siglec-7 GT1b 1.3 Simian virus GM1 3.2 VP1Tetanus toxin GT2 1.2 HCR/T GLIDE Siglec-7 GT1b 9.6 Simian virus GM1Unable to VP1 find docked pose Tetanus toxin GT2 4.2 HCR/T

EXAMPLE 12

Improving Effector Functions by Enhancing Affinities for Antigen and FcReceptor (FcR)

Anti-GD2 MoAb is a proven therapy for chemo-resistant metastaticneuroblastoma (NB), with ch14.18 providing a benchmark for nextgeneration MoAb. The combination of anti-GD2 MoAb mouse 3F8 (m3F8) andgranulocyte-macrophage colony-stimulating factor (GM-CSF) hasconsistently induced complete marrow remission in 80% of patients withprimary refractory NB. Among patients treated in first complete/verygood partial remission (CR/VGPR), this combination plus 13-cis-Table 12.3F8 amino acids residues that directly interact with GD2 based onCDOCKER and CHARMm energy minimization retinoic acid improved PFS to51%±7% and OS to 80%±5% past 5 years. Outcome analyses have repeatedlyshown the importance of FcR affinity based on gene polymorphisms in MoAbtherapy of NB and other solid tumors. Improving affinities for antigenand for FcR without sacrificing specificity should improve MoAb efficacyin vitro and in vivo.

TABLE 12 3F8 amino acid residues that directly interact with GD2 basedon CDOCKER and CHARMm energy minimization Amino Acid and Chain PositionLight chain Asp 91 Tyr 92 Heavy chain Gly 33 Trp 52 Ala 53 Gly 54 Gly 55Ile 56 Asn 58 Arg 98 His 101 Tyr 102 Gly 103 Tyr 104

TABLE 13 Top CDR mutants from in silico mutational analysis of 3F8interacting residues with GD2 head group Change in interaction energyWildtype Mutation (kcal/mol) HC GLY54 ILE −8.23 HC GLY103 LEU −2.38 HCGLY103 TRP −1.9 HC GLY55 THR −1.38 HC ILE56 ARG −0.93 HC GLY54 SER −0.86

Methods:

Chimeric 3F8 (ch3F8) and humanized 3F8 (hu3F8) of the huIgG1 subclasswere constructed by genetic engineering, expressed in CHO cells andpurified by protein A affinity chromatography. An Fc-glycoform of hu3F8(hu3F8n or hu3F8-MAGE1.5) that lacked fucose and N-acetylglucosamine wasproduced in special MAGE1.5 CHO cells. Affinities of these MoAb formsfor GD2 and for FcR were compared using BIACORE T-100. Effectorfunctions were tested using antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) assays,and their potencies derived from EC50 of MoAb. These strategies wereapplied to MoAb against other tumor antigen systems (B7-H3, HER-2,CSPG4, LI CAM) with clinical potential.

Results:

Ch3F8 and hu3F8 maintained a Kd similar to that of m3F8, all sharing a10-fold slower koff, when compared to 14G.2a or ch14.18, resulting in amore favorable KD and longer residence time. M3F8, ch3F8 or hu3F8inhibited proliferation of NB cell lines in vitro, while other anti-GD2antibodies were ineffective. In peripheral blood mononuclear cell(PBMC)-ADCC, granulocyte (PMN)-ADCC, or CMC, ch3F8 and hu3F8 were morethan 10-fold stronger than ch14.18. PBMC-ADCC by hu3F8n was 10 fold moreefficient than hu3F8, and >100 fold more potent than m3F8. While¹³¹I-hu3F8 in biodistribution studies showed tumor to normal tissueratios comparable to those of ¹³¹I-m3F8, hu3F8 demonstrated superioranti-tumor effect against NB xenografts. Although similar conclusionscould be drawn for chimeric and humanized antibodies specific for othertumor antigen systems, the nature of the tumor epitope/antigen wascritical in determining the efficiency of tumor kill.

Conclusions:

While koff (or K_(D)) for antigen or FcR individually improved effectorfunctions, together their effects appeared multiplicative for anti-GD2MoAb 3F8. Future strategies directed at improving both affinities shouldfurther extend the clinical efficacy of MoAb observed so far.

EXAMPLE 13

Further Fc Modification to Improve FcR Binding:

Hu3F8-IgG1-DEL with the triple mutation DEL (S239D/A330L/I332E)(Lazar etal., 2006, PNAS USA 103, 4005-10) in the heavy chain have been made withsubstantial increase in affinity to FcR3A and FcR2A, but also to FcR2B(see BIACORE data in table 14). However, the A/I ratio for activatingversus inhibitory signals for hu3F8-IgGn is 196 for CD16-158V, comparedto 30 for hu3F8-IgG1-DEL, which has been postulated to have clinicaladvantage (Nimmerjahn and Ravetch, Immunol Rev, 236:265-275, 2010).

TABLE 14 Relative affinity. K_(D) was determined by flowing antibodiesover recombinant FcR on CM5 chip using BIACORE T-100. All values werenormalized to hu3F8-IgG1 binding on FcRIII-158F. FcRIII FcRII Antibodyform 158V 158F CD16B 131H 131R CD32B hu3F8-IgG1 2.5 1.0 0.8 2.1 1.0 0.1hu3F8-IgG1-DEL 35.9 24.2 24.1 2.8 2.0 1.2 hu3F8-IgG1n 19.6 6.0 2.3 1.20.4 0.1

CD16 Mediated ADCC on Neuroblastoma LAN-1.

NK92-CD16 effector cells were added to LAN1 neuroblastoma tumor targetsat effector:target ratio of 5:1 at different antibody concentrations ina 4 hour ⁵¹Cr release assay.

The increase in affinity for FcR translated into increase in ADCCpotency mediated by CD16 or CD32. For ADCC mediated by CD16 (data notshown), hu3F8-IgG1-DEL and hu3F8-IgG1n were equivalent.

Complement Mediated Lysis of Neuroblastoma LAN-1.

Human complement was added to neuroblastoma LAN-1 tumor targets at afinal serum complement dilution of 1:100, at increasing antibodyconcentrations in a 4 hour ⁵¹Cr release assay. In complement mediatedlysis (CMC, data not shown), hu3F8-IgG1-DEL was not worse thanhu3F8-IgG1 or hu3F8-IgG1n. When triple mutation in IgG heavy chain wascombined with hu3F8-IGg1n, there was no additional improvement in ADCC.

EXAMPLE 15

Arming T Cells and Effectors In Vivo

T cells or T lymphocytes are WBC that are key players in cell-mediatedimmunity. They can be distinguished from other lymphocyte types, such asB cells and natural killer (NK) cells by the presence of a specialreceptor on their cell surface called T cell receptors (TCR). They alsocarry a unique surface marker called CD3. T stands for thymus, which isthe principal organ responsible for the maturation of T cells. Severalsubsets of T cells exist, each with a distinct function. T helper cells(TH cells) assist other WBC in immunologic processes, includingmaturation of B cells to secrete antibodies and activation of cytotoxicT cells and macrophages. They have come to be called CD4+ T cellsbecause they carry the CD4 protein. When activated, Helper T cellsdivide rapidly and secrete cytokines to regulate or assist the immuneresponse. These cells can differentiate into several subtypes (e.g. TH1,TH2, TH3, TH17, or TFH) secreting different cytokines to modulate theimmune response. In some tumor models, CD4+ T cells are also sufficientin suppressing cancer growth. Although the regular activation signalscome from the antigen presenting cells (APC), CD4+ T cells can becomeactivated when their surface CD3 is cross linked by antibodies.Cytotoxic T cells (CTLs) destroy virally infected cells and tumor cells,and also responsible for transplant or graft rejection. They are calledCD8+ T cells because they express the surface CD8 glycoprotein. Theyengage targets by recognizing antigens associated with majorhistocompatibility complex (MHC) class I molecules, which are absent orlow on many human tumors. Memory T cells are antigen-specific T cellsthat persist for a long time after the virus or the tumor cells arekilled. They can quickly expand to large numbers when challenged withthe tumor antigen, thus empowering the immune system with “memory”against cancers. Natural killer T cells (NKT cells) are a special kindof T-cells that bridges the adaptive immune system with the innateimmune system. Unlike conventional T cells that recognize peptideantigen on the MHC, NKT cells recognize glycolipid antigen presented bya molecule called CD1d. Once activated, these cells can performfunctions similar to both Th and Tc cells (i.e., cytokine production andrelease of cytolytic/cell killing molecules). They are known torecognize and eliminate tumor cells. γδT cells (gamma delta T cells)represent a small subset of T cells that carry a TCR on their surface. Amajority of T cells have a TCR composed of two glycoprotein chainscalled α-and β-TCR chains. However, in γδT cells, the TCR is made up ofone γ-chain and one δ-chain. They represent only 5% of total T cells,but are most abundant in the gut mucosa (and named intraepitheliallymphocytes, IELs). The antigens that activate γδT cells are not known;however, γδT cells are not MHC restricted, and probably recognize wholeproteins rather than peptides on MHC.

Adoptive immunotherapy trials in 1986 using lymphokine-activated killercells (LAK) and tumor infiltrating lymphocytes (TIL) reported occasionaltumor responses in patients. Donor lymphocyte infusions have shown evenmore successes in patients with chronic myelogenous leukemia followingallogeneic stem cell transplant or in patients with post-transplantEBV-associated lymphoproliferative disease (PTLD). In solid tumors, CTLwas successful in treating malignant melanoma during the lymphopenicphase created by high dose chemotherapy. Bispecific antibodies are madeby fusing two hybridomas to create hybrid immunoglobulin molecules withtwo binding sites. The antibodies not only handcuff tumors to T-cells;they cross-link CD3 on T-cells and initiate the activation cascade. Thisway, TCR-based cytotoxicity is redirected to desired tumor targetsbypassing MHC restrictions. Arming of polyclonally activated T cells(ATC) with anti-CD3×anti-TAA (BsAb or BiTE antibody) combines thetargeting specificity of MoAb (e.g. hu3F8 where TAA is GD2) with thenon-MHC-restricted perforin/granzyme mediated cytotoxicity of T cells.BsAb or BiTE can arm ex vivo expanded activated T cells before infusioninto a patient. This strategy converts every ATC into a specific CTL(Thakur and Lum, 2010, Curr Opin Mol Ther 12, 340-349; Grabert et al.,2006, Clin Cancer Res 12, 569-576).

Tumors evade T cells by a number of mechanisms: low or no expression ofMHC (e.g. in NB), derailing T cell signaling, decreased presentation oftumor peptides on MHC, absence of co-stimulatory molecules, andinduction of regulatory T-cells that inhibit CTL and humoral responses.Since the killing carried out by BsAb or BiTE armed ATC isnon-MHC-restricted, this strategy should overcome some of these tumorescape mechanisms. Tumors secrete TGF-β shifting the T-cell immuneresponse to a Th2 type, downregulating interleukin 2 (IL-2) and IFN-γsecretion, while upregulating IL-10 and IL-6, all leading to immunesuppression. T-cells redirected by BsAb or BiTE may bypass thesenegative effects of regulatory cytokines, since armed ATC lyse tumortargets in an IL-2 independent manner. Patients treated with BsAb orBiTE armed T cells directed at their tumors have increased levels ofTNF-α and IFN-γ, which should shift the T-cells towards a Th1 response.In addition, cytotoxic T cells kill through their Fas ligand (FasL) thatengage Fas receptors (CD95) on tumor cells. Unfortunately, FasL ontumors cells can also induce apoptosis of T cells. TCR stimulationthrough CD3 cascade protects CD8+ cells from CD95-mediated suicide.Armed ATC resist CD95-induced cell death through crosslinking of the TCRwith BsAb or BiTE. The ability of T-cells to kill serially, i.e. oneT-cell killing consecutive tumor targets, proliferate during theprocess, and move into lymphatics and soft tissues increased the chanceof catching NB cells while they metastasize out of the marrow space toform tumor masses. Recent studies using BsAb or BITE targeting humancancers have shown promise.

There is mounting evidence, particularly from analyses of patients whohave received allogenic hematopoietic cell transplants, supporting thepotential of T-cells to suppress or eradicate lymphomas and certainforms of leukemia (O'reilly et al., 2010, Semin Immunol 22, 162-172).However, there are no convincing data supporting a role for T-cells inthe control of solid tumors in children. This is consistent with thefact that severed of these tumors either do not express inherited classI or II HLA alleles (e.g. neuroblastoma) (Raffaghello et al., 2005,Oncogene 24, 4634-4644; Wolfl et al., 2005, Cancer Immunol Immunother54, 400-406) or express only class I alleles and at low levels (e.g.rhabdomyosarcomas) (Prados et al., 2006, Neoplasma 53, 226-231).Furthermore, expression of critical costimulatory molecules such as B7.1and ICAM-1 is often low or undetectable. As a result, the capacity ofthese tumors to elicit T-cell responses is poor and the potential ofeffector T-cells to engage the tumors through T-cell receptor by bindingtumor antigens presented by HLA alleles is limited. Furthermore, themost effective therapies currently available for neuroblastoma,rhabdomyosarcoma, Ewing's sarcoma and desmoplastic small round celltumors employ immunosuppressive alkylating agents, particularlycyclophosphamide at doses inducing profound T-lymphopenia. Bifunctionalantibodies permit the targeted engagement of T-cells and exploitation oftheir effector functions through HLA-non-restricted CD3-mediatedactivation rather than their antigen-specific HLA-restricted TCRs.Studies of certain bifunctional monoclonal antibodies specific for CD3and a tumor antigen such as CD-19, HER-2 NEU, or CEA have demonstratedthe capacity of these antibodies to link cytotoxic T-cells to tumorcells expressing the other targeted antigen (Bargou et al., 2008,Science 321, 974-977; Topp et al., 2009, Blood (ASH Annual MeetingAbstracts) 114, 840; Kiewe et al., 2006, Clin Cancer Res 12, 3085-3091;Lutterbuese et al., 2009, J Immnother 32, 341-352). Once both antibodyreceptors are engaged, a cytotoxic T-cell response is initiated againstthe tumor cells. The T-cell response involves formation of a cytotoxicsynapse between the T-cell receptor and the tumor cell as well asperforin and granzyme mediated induction of tumor cell apoptosis (Offneret al., 2006, Mol Immunol 43, 763-771; Brischwein et al., 2006, MolImmunol 43, 1129-1143). Engagement of CD3 also activates the T-cells,inducing proliferation and generation of effector cytokines thatpotentiate the antitumor effect (Brischwein et al., 2006, supra;Brischwein et al., 2007, J Immunother 30, 798-807). Strikingly, theactivated T-cells upregulate an anti-apoptotic protein c-FLIP whichprotects them from the cytotoxic effects of TNF and Fas ligand generatedduring T-cell activation (Dreir et al., 2002, Int J Cancer 100,690-697). As a result, the T-cell response is magnified. As aconsequence, picogram levels of the bifunctional antibody can exertsignificant antitumor effects in vitro (Lutterbuese et al., 2009, supra;Brandl et al., 2007, Cancer Immunol Immunother 56, 1551-1563) and invivo, as shown in preclinical animal models and particularly in theresults of initial clinical trials of the CD3/CD19 bispecific in thetreatment of B-cell lymphomas and ALL (Topp et al., 2009, supra; Kieweet al., 2006, supra). It has been hypothesized that the T-cell responsesinduced can also recruit naïve T-cells and stimulate the generation oftumor-specific T-cells at tumor sites (Koehne et al., 2002, Blood 99,1730-1740). Bispecific antibodies can also be used to retarget othereffector cells besides T-lymphocytes. These effector cells include NKcells, B-lymphocytes, dendritic cells, monocytes, macrophages,neutrophils, mesenchymal stem cells, neural stem cells and other stemcells to cells, tissues or organs that express GD2. When the tissue istumor, these effector cells can be exploited to kill or to depositproteins (e.g. cytokines, antibodies, enzymes, or toxins), radioactiveisotopes for diagnosis or for therapy. When the tissue is a normalorgan, the effector cells can be similarly exploited to deliver proteinsor isotopes for diagnosis or for therapy.

Bispecific MoAb may be comprised of dual variable domains, with onedomain having anti-3F8 variable domain and the other domain chosen froma group consisting of anti-OKT3 for retargeting T cells for tumorcytotoxicity, or DOTA-metal, C8.2.5 for multistep pretargeting, or Clone35, CD137, for ADCC with anti-41BB-scFv as agonist, or with CD137, 41BBLfor ADC with 41 BBL as agonist. A N297A mutation in the CH2 domainresults in aglycosylation leading to no FcR or C1q binding. The aminoacid sequence of (hu3F8-LC)-(huOKT3-scFv) where the huOKT3-scFv isdisulfide stabilized is shown in SEQ ID NO:23. The amino acid sequenceof hu3F8-LC)-(C8.2.5-scFv) (based on Orcutt et al., 2010, Protein EngDesign and Selection 23, 221) with C8.2.5-scFv disulfide stabilized isshown in SEQ ID NO:24.

Bispecific antibody (anti-GD2 and anti-DOTA) can be used in a first stepof a multistep pretargeting, followed by blood clearance usingDOTA(metal)-Dextran as clearing agent, with a third step introducingDOTA(metal)-conjugated therapeutics such as DOTA(metal)-radioactivemetal, DOTA(metal)-nanoparticles, DOTA(metal-liposomes,DOTA(metal)-drugs,

DOTA(metal)-DNA, DOTA(metal)-RNA, and DOTA(metal)-toxins. Since C8.2.5has different affinities for each type of DOTA-metal comples, theaffinity of the pretargeted C8.2.5 for the clearing agent and theDOTA-ligand can be precisely controlled.

The amino acid sequence of 3F8, hu3F8 and its variants, can be used toconstruct chimeric antigen receptor (CAR) as was previously shown forother anti-GD2 antibodies (Krause et al., 1998, J Exp Med 188, 619-626).The CAR strategy of retargeting immune effector cells is independent ofthe MHC-peptide-TCR interaction and allows cells to react against alarge variety of cell surface antigens (Davies and Maher, 2010, Achivumimmunologiae et therapiae experimentalis 58, 165-178). Several methodshave been used in the design of CARs, with most of them employing theantigen binding domain of a monoclonal antibody in the form of asingle-chain variable fragment (scFv) for antigen recognition. Theinitial T cell activating receptors originated from studies whichallowed researchers to elucidate the role of the CD3ζ chain (Irving andWeiss, 1991, Cell 64, 891-901; Romeo et al., 1992, Cell 68, 889-897). Insubsequent studies, scFvs of interest were fused to the CD3ζ chain(Eshhar et al., 1993, PNAS USA 90, 720-724) or FcERIγ (Weijtens et al.,1996, J Immunol 157, 836-843), and both were found to be sufficient forT cell activation. While this laid the blueprint for CAR construction,the incorporation of costimulatory molecules came about after it wasfound that first generation CARs were able to induce T cellproliferation only up to 2-3 cell divisions, followed rapidly by celldeath (Gong et al., 1999, Neoplasia 1, 123-127). By expressing CD80 onthe target tumor cell, researchers were able to show that CAR expressingcells could be restimulated, leading to further increases in T cellnumbers. The first CARs which incorporated the CD28 costimulatorymolecule alongside the CD3ζ chain showed vast improvements over thosewhich expressed the CD3ζ chain alone (Krause et al., 1998, supra; Hayneset al., 2002, Blood 100, 3155-3163; Maher et al., 2002, Nature Biotech20, 70-75); this included an absolute increase in T cell numbers as wellas an increase in IL-2 production. Since then, several other groupsbegan to use other costimulatory molecules, either in combination withCD3ζ alone or with both CD3ζ and CD28. These additional signalingmolecules include 4-1BB (Wang et al., 2007, Human Gene Ther 18, 712-725;Brentjens et al., 2007, Clin Cncer Res 13, 5426-5432; Imai et al., 2004,Leukemia 18, 676-684; Finney et al., 2004, J Immunol 172, 104-113),DAP10 (Brentjens et al., 2007, supra), OX40 (Brentjens et al., 2007,supra; Finney et al., 2004, supra; Wilkie et al., 2008, J Immunol 180,4901-4909; Nguyen and Geiger, 2003, Gene Therapy 10, 594-604; Pule etal., 2005, Mol Ther 12, 933-941) and ICOS (Finney et al.,2004, supra),and have been applied in the context of T cells as well as NK cells(Daldrup-Link et al., 2005, Eropean radiology 15, 4-13; Imai andCampana, 2004, J Biol Reg Homeostatic Ag 18, 62-71; Roberts et al.,1998, J Immunol 375-384; Kruschinski et al., 2008, PNAS USA 105,17481-17486; Pegram et al., 2008, J Immunol 181, 3449-3455). While firstgeneration CARs are the only ones which have been tested in the clinicup to this point, both in vitro and in vivo comparisons havedemonstrated a clear superiority with second and third generation CARs(Haynes et al., 2002, supra; Brentjens et al., 2007, supra; Teng et al.,2004, Human Gene Ther 15, 699-708; Haynes et al., 2002, J Immunol 169,5780-5786; Kowolik et al., 2006, Cancer Res 66, 10995-11004; Loskog etal., 2006, Leukemia 20, 1819-1928; Moeller et al., 2004, Cancer GeneTherapy 11, 371-379; Vera et al., 2006, Blood 108, 3890-3897).

Currently, most researchers use bulk human peripheral T cells, howeverothers have recently began to use EBV-specific T cells (Rossig et al.,2002, Blood 99, 2009-2016), lymphoid progenitor cells (Zakrzewski etal., 2006, Nature Med 12, 1039-1047; Zakrzewski et al., 2008, NatureBiotech 26, 453-461), and unfractionated bone marrow cells (Papapetrouet al., 2009, J clin Invest 119, 157-168; Wang et al., 1998, Nature Med4, 168-172). Killer leukemia cell lines (e.g. NK92, NK92MI, KHYG-1) thatare cytolytic and easy to culture can also provide a continuous supplyof CAR expressing effector cells for pre-clinical and clinical testing.NK92MI is a human NK cell line derived from a non-Hodgkin's lymphoma andtransduced with human IL-2 cDNA; previous studies have demonstrated itsstrong cytotoxic abilities in mouse models (Tam et al., 1999, J Hematol8, 281-290; Korbelik and Sun, 2001, Inter J Cancer 93, 269-274). Inaddition, NK92 cells have also been used in the clinical setting andproven safe after a number of Phase I studies in patients with renalcell carcinoma and melanoma (Arai et al., 2008, Cytotherapy 10,625-632). Because of their ease of maintenance in vitro and relativelyshort doubling-times, these cells are ideal effectors for variouscytotoxicity assays to test a variety of targeting approaches. Whilestudies using the original IL-2-dependent NK92 cell line have shownminimal toxicities in both mice and humans, the IL-2-transduced NK92MIcells may have a greater leukemogenic potential. One method by whichresearchers try and avoid leukemogenesis in SCID mice using NK92 cellsis by irradiating the effectors with 3000 cGy before inoculation. Inphase I clinical trials, this is sufficient in preventing NK92MI cellsfrom proliferating uncontrollably inside of the immunocompromisedpatient. An alternative safety mechanism is that which involves theemployment of suicide genes. One common example is the use of theherpesvirus thymidine kinase gene, which works by killing a cell whichexpresses the gene by administration of acyclovir or ganciclovir (Heleneet al., 1997, J Immunol 5079-5082).

1-36. (canceled)
 37. A humanized or chimeric antibody or fragment thereof capable of binding to GD2, wherein the antibody or fragment thereof comprises any of the following: (i) a variable heavy chain domain of SEQ ID NO: 1 and a variable light chain domain of SEQ ID NO:2; (ii) a variable heavy chain domain of SEQ ID NO:3 and a variable light chain domain of SEQ ID NO:2; (iii) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO:5; (iv) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO:7; (v) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:5; (vi) a variable heavy chain domain of SEQ ID NO:9 and a variable light chain domain of SEQ ID NO: 10; and (vii) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO:
 5. 38. An isolated nucleic acid molecule encoding a humanized or chimeric antibody or fragment thereof capable of binding GD2, wherein the humanized or chimeric antibody or fragment thereof comprises any of the following: (i) a variable heavy chain domain of SEQ ID NO: 1 and a variable light chain domain of SEQ ID NO:2; (ii) a variable heavy chain domain of SEQ ID NO:3 and a variable light chain domain of SEQ ID NO:2; (iii) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO:5; (iv) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO:7; (v) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:5; (vi) a variable heavy chain domain of SEQ ID NO:9 and a variable light chain domain of SEQ ID NO: 10; (vii) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO: 5; (viii) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO: 7; and (ix) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:
 7. 39. A method of treating or preventing a medical condition in the subject, comprising administering a therapeutically effective amount of a humanized or chimeric antibody or fragment thereof, wherein the humanized or chimeric antibody or fragment thereof comprises any of the following: (i) a variable heavy chain domain of SEQ ID NO: 1 and a variable light chain domain of SEQ ID NO:2; (ii) a variable heavy chain domain of SEQ ID NO:3 and a variable light chain domain of SEQ ID NO:2; (iii) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO:5; (iv) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO:7; (v) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:5; (vi) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO: 7; (vii) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO: 5; (viii) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO: 7; and (ix) a variable heavy chain domain of SEQ ID NO:9 and a variable light chain domain of SEQ ID NO:
 10. 40. The method of claim 39, wherein the medical condition is characterized by GD2 expression.
 41. The method of claim 39, wherein the medical condition is selected from the group consisting of: a neuroblastoma, a melanoma, a sarcoma, a brain tumor and a carcinoma.
 42. The method of claim 40, wherein the medical condition is a GD2-positive tumor.
 43. The method of claim 40, wherein the medical condition is selected from the group consisting of: osteosarcoma, liposarcoma, fibrosarcoma, malignant fibrous, histiocytoma, leimyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, HTLV-1 infected T cell leukemia.
 44. The method of claim 39, wherein the humanized or chimeric antibody or fragment is glycosylated with terminal mannose, N-acetylglucose or glucose, but no fucose.
 45. The method of claim 39, wherein the humanized or chimeric antibody comprises a variant Fc region.
 46. The method of claim 45, wherein the variant Fc region comprises a substitution of S239D, A330L and 1332E.
 47. The method of claim 45, wherein the variant Fc region comprises a N297A substitution.
 48. The method of claim 39, wherein the humanized or chimeric antibody has an altered affinity for an FcγR.
 49. The method of claim 48, wherein the humanized or chimeric antibody has increased affinity for an FcγR as compared to a reference humanized or chimeric antibody.
 50. The method of claim 48, wherein the humanized or chimeric antibody is characterized by about 10-fold higher antibody-dependent cellular cytotoxicity (ADCC) as compared to a reference humanized or chimeric antibody.
 51. The method of claim 48, wherein the humanized or chimeric antibody is characterized by about 100-fold higher antibody-dependent cellular cytotoxicity (ADCC) as compared to a reference humanized or chimeric antibody.
 52. The method of claim 48, wherein the humanized or chimeric antibody is characterized by an activating:inhibiting FcγR receptor ratio of about 100 or more as compared to a reference humanized or chimeric antibody.
 53. The method of claim 39, wherein the step of administering comprises administering a pharmaceutical composition that delivers the humanized or chimeric antibody or fragment thereof, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent.
 54. The method of claim 53, wherein the pharmaceutical composition comprises the humanized or chimeric antibody or fragment thereof at a concentration of 0.1 mg/ml to 10 mg/ml.
 55. The method of claim 53, wherein the pharmaceutical composition comprises a citrate buffer.
 56. The method of claim 54, wherein the pharmaceutical composition has a pH of 6.0 to 8.0.
 57. The method of claim 54, wherein the pharmaceutical composition comprises 1-10% human serum albumin.
 58. The method of claim 53, wherein the pharmaceutical composition that delivers the humanized or chimeric antibody or fragment thereof is administered at a dose of 0.5 to 10 mg/kg.
 59. The method of claim 53, wherein the pharmaceutical composition that delivers the humanized or chimeric antibody or fragment thereof is administered at a dose of 2 to 4 mg/kg.
 60. The method of claim 53, wherein the step of administering comprises administering a plurality of doses.
 61. The method of claim 53, wherein the step of administering comprises administering intravenously.
 62. The method of claim 53, wherein the step of administering comprises administering by infusion.
 63. The method of claim 53, wherein the step of administering comprises administering multiple cycles of the composition.
 64. The method of claim 39, wherein the step of administering comprises administering to a subject who has received or is receiving granulocyte-macrophage colony-stimulating factor (GM-CSF)
 65. The method of claim 39, wherein the step of administering comprises administering to a subject who has received premedication with one or more of analgesics, antihistamines, and 5-minute infusions of 2 mg/m2 HM3F8.
 66. A method of treating or preventing a medical condition in the subject, comprising administering a therapeutically effective amount of a nucleic acid molecule encoding a bispecific antibody having first and second antigen-binding sites, which first antigen-binding site comprises a light chain and a heavy chain of a humanized 3F8 antibody, wherein the humanized 3F8 antibody comprises any of the following: (i) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO:5; (ii) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO:5; (iii) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:5; (iv) a variable heavy chain domain of SEQ ID NO:9 and a variable light chain domain of SEQ ID NO: 10; (v) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO: 7; (vi) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO: 7; and (vii) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:
 7. 67. The nucleic acid molecule of claim 66, wherein the second antigen-binding site is specific for CD3 or for a DOTA (metal).
 68. The nucleic acid molecule of claim 67, wherein the second antigen-binding site comprises a peptide of SEQ ID NO:23 that includes the second antigen-binding site.
 69. The nucleic acid molecule of claim 68, wherein the second antigen-binding site comprises a peptide of SEQ ID NO:24 that includes the second antigen-binding site.
 70. The method of claim 66, wherein the medical condition is characterized by GD2 expression.
 71. The method of claim 66, wherein the medical condition is selected from the group consisting of: a neuroblastoma, a melanoma, a sarcoma, a brain tumor and a carcinoma.
 72. The method of claim 70, wherein the medical condition is a GD2 positive tumor.
 73. The method of claim 70, wherein the medical condition is selected from the group consisting of: osteosarcoma, liposarcoma, fibrosarcoma, malignant fibrous, histiocytoma, leimyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, HTLV-1 infected T cell leukemia.
 74. The method of claim 66, wherein the step of administering comprises administering a pharmaceutical composition that delivers the nucleic acid, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent.
 75. The method of claim 74, wherein the step of administering comprises administering a plurality of doses.
 76. The method of claim 74, wherein the step of administering comprises administering intravenously.
 77. The method of claim 74, wherein the step of administering comprises administering by infusion.
 78. The method of claim 74, wherein the step of administering comprises administering multiple cycles of the composition. 